Compositions and methods for modulating angiopoietin-like 3 expression

ABSTRACT

Provided herein are methods, compounds, and compositions for reducing expression of an ANGPTL3 mRNA and protein in an animal. Also provided herein are methods, compounds, and compositions for reducing lipids and/or glucose in an animal. Such methods, compounds, and compositions are useful to treat, prevent, delay, or ameliorate any one or more of cardiovascular disease and/or metabolic disease, or a symptom thereof, in an individual in need thereof.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledBIOL0254USC2SEQ_ST25.txt, created on Feb. 26, 2018 which is 0.98 MB insize. The information in the electronic format of the sequence listingis incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

Provided herein are methods, compounds, and compositions for reducingexpression of angiopoietin-like 3 (ANGPTL3) mRNA and protein in ananimal. Also, provided herein are methods, compounds, and compositionshaving an ANGPTL3 inhibitor for reducing ANGPTL3 related diseases orconditions in an animal. Such methods, compounds, and compositions areuseful, for example, to treat, prevent, delay or ameliorate any one ormore of cardiovascular disease or metabolic syndrome, or a symptomthereof, in an animal.

BACKGROUND

Diabetes and obesity (sometimes collectively referred to as “diabesity”)are interrelated in that obesity is known to exacerbate the pathology ofdiabetes and greater than 60% of diabetics are obese. Most human obesityis associated with insulin resistance and leptin resistance. In fact, ithas been suggested that obesity may have an even greater impact oninsulin action than diabetes itself (Sindelka et al., Physiol Res.,2002, 51, 85-91). Additionally, several compounds on the market for thetreatment of diabetes are known to induce weight gain, a veryundesirable side effect to the treatment of this disease.

Cardiovascular disease is also interrelated to obesity and diabetes.Cardiovascular disease encompasses a wide variety of etiologies and hasan equally wide variety of causative agents and interrelated players.Many causative agents contribute to symptoms such as elevated plasmalevels of cholesterol, including non-high density lipoproteincholesterol (non-HDL-C), as well as other lipid-related disorders. Suchlipid-related disorders, generally referred to as dyslipidemia, includehyperlipidemia, hypercholesterolemia and hypertriglyceridemia amongother indications. Elevated non-HDL cholesterol is associated withatherogenesis and its sequelae, including cardiovascular diseases suchas arteriosclerosis, coronary artery disease, myocardial infarction,ischemic stroke, and other forms of heart disease. These rank as themost prevalent types of illnesses in industrialized countries. Indeed,an estimated 12 million people in the United States suffer with coronaryartery disease and about 36 million require treatment for elevatedcholesterol levels.

Epidemiological and experimental evidence has shown that high levels ofcirculating triglyceride (TG) can contribute to cardiovascular diseaseand a myriad of metabolic disorders (Valdivielso et al., 2009,Atherosclerosis Zhang et al., 2008, Circ Res. 1; 102(2):250-6). TGderived from either exogenous or endogenous sources is incorporated andsecreted in chylomicrons from the intestine or in very low densitylipoproteins (VLDL) from the liver. Once in circulation, TG ishydrolyzed by lipoprotein lipase (LpL) and the resulting free fattyacids can then be taken up by local tissues and used as an energysource. Due to the profound effect LpL has on plasma TG and metabolismin general, discovering and developing compounds that affect LpLactivity are of great interest.

Metabolic syndrome is a combination of medical disorders that increaseone's risk for cardiovascular disease and diabetes. The symptoms,including high blood pressure, high triglycerides, decreased HDL andobesity, tend to appear together in some individuals. It affects a largenumber of people in a clustered fashion. In some studies, the prevalencein the USA is calculated as being up to 25% of the population. Metabolicsyndrome is known under various other names, such as (metabolic)syndrome X, insulin resistance syndrome, Reaven's syndrome or CHAOS.With the high prevalence of cardiovascular disorders and metabolicdisorders there remains a need for improved approaches to treat theseconditions

The angiopoietins are a family of secreted growth factors. Together withtheir respective endothelium-specific receptors, the angiopoietins playimportant roles in angiogenesis. One family member, angiopoietin-like 3(also known as angiopoietin-like protein 3, ANGPT5, ANGPTL3, orangiopoietin 5), is predominantly expressed in the liver, and is thoughtto play a role in regulating lipid metabolism (Kaplan et al., J. LipidRes., 2003, 44, 136-143). Genome-wide association scans (GWAS) surveyingthe genome for common variants associated with plasma concentrations ofHDL, LDL and triglyceride found an association between triglycerides andsingle-nucleotide polymorphisms (SNPs) near ANGPTL3 (Willer et al.,Nature Genetics, 2008, 40(2):161-169). Individuals with homozygousANGPTL3 loss-of-function mutations present with low levels of allatherogenic plasma lipids and lipoproteins, such as total cholesterol(TC) and TG, low density lipoprotein cholesterol (LDL-C), apoliprotein B(apoB), non-HDL-C, as well as HDL-C (Romeo et al. 2009, J Clin Invest,119(1):70-79; Musunuru et al. 2010 N Engl J Med, 363:2220-2227;Martin-Campos et al. 2012, Clin Chim Acta, 413:552-555; Minicocci et al.2012, J Clin Endocrinol Metab, 97:e1266-1275; Noto et al. 2012,Arterioscler Thromb Vasc Biol, 32:805-809; Pisciotta et al. 2012,Circulation Cardiovasc Genet, 5:42-50). This clinical phenotype has beentermed familial combined hypolipidemia (FHBL2). Despite reducedsecretion of VLDL, subjects with FHBL2 do not have increased hepatic fatcontent. They also appear to have lower plasma glucose and insulinlevels, and importantly, both diabetes and cardiovascular disease appearto be absent from these subjects. No adverse clinical phenotypes havebeen reported to date (Minicocci et al. 2013, J of Lipid Research,54:3481-3490). Reduction of ANGPTL3 has been shown to lead to a decreasein TG, cholesterol and LDL levels in animal models (U.S. Ser. No.13/520,997; PCT Publication WO 2011/085271). Mice deficient in ANGPTL3have very low plasma triglyceride (TG) and cholesterol levels, whileoverpexpression produces the opposite effects (Koishi et al. 2002;Koster 2005; Fujimoto 2006). Accordingly, the potential role of ANGPTL3in lipid metabolism makes it an attractive target for therapeuticintervention.

To date, therapeutic strategies to treat cardiometabolic disease bydirectly targeting ANGPTL3 levels have been limited. ANGPTL3 polypeptidefragments (U.S. Ser. No. 12/128,545), anti-ANGPTL3 antibodies (U.S. Ser.No. 12/001,012) and ANGPTL3 nucleic acid inhibitors including antisenseoligonucleotides (U.S. Ser. No. 13/520,997; PCT Publication WO2011/085271; incorporated by reference herein, in their entirety) havepreviously been suggested or developed, but none of the compoundsdirectly targeting ANGPTL3 have been approved for treatingcardiometabolic disease. Accordingly, there is an unmet need for highlypotent and tolerable compounds to inhibit ANGPTL3. The inventiondisclosed herein relates to the discovery of novel, highly potentinhibitors of ANGPTL3 expression and their use in treatment.

SUMMARY OF THE INVENTION

Provided herein are compositions and methods for modulating expressionof ANGPTL3 mRNA and protein. In certain embodiments, the composition isan ANGPTL3 specific inhibitor. In certain embodiments, the ANGPTL3specific inhibitor decreases expression of ANGPTL3 mRNA and protein.

In certain embodiments, the composition is an ANGPTL3 specificinhibitor. In certain embodiments, the ANGPTL3 specific inhibitor is anucleic acid. In certain embodiments, the nucleic acid is an antisensecompound. In certain embodiments, the antisense compound is a modifiedoligonucleotide. In certain embodiments, the antisense compound is amodified oligonucleotide with a conjugate group attached.

In certain embodiments, the ANGPTL3 specific inhibitor is a modifiedoligonucleotide with a conjugate group, wherein the modifiedoligonucleotide consists of 12 to 30 linked nucleosides and having anucleobase sequence comprising at least 8, least 9, least 10, least 11,at least 12, least 13, at least 14, at least 15, at least 16, least 17,least 18, least 19, or 20 contiguous nucleobases of the nucleobasesequence of SEQ ID NO: 77.

In certain embodiments, the ANGPTL3 specific inhibitor is a modifiedoligonucleotide with a conjugate group, wherein the modifiedoligonucleotide consists of 12 to 30 linked nucleosides and comprising anucleobase sequence comprising a portion of at least 8 contiguousnucleobases complementary to an equal length portion ofnucleobases1140-1159 of SEQ ID NO: 1, wherein the nucleobase sequence of themodified oligonucleotide is at least 80% complementary to SEQ ID NO: 1.

In certain embodiments, the ANGPTL3 specific inhibitor is a modifiedoligonucleotide with a conjugate group, wherein the modifiedoligonucleotide consists of 12 to 30 linked nucleosides and comprising anucleobase sequence comprising a portion of at least 8 contiguousnucleobases complementary to an equal length portion of nucleobases9715-9734 of SEQ ID NO: 2, wherein the nucleobase sequence of themodified oligonucleotide is at least 80% complementary to SEQ ID NO: 2.

In certain embodiments, the ANGPTL3 specific inhibitor is a modifiedoligonucleotide with a conjugate group, wherein the modifiedoligonucleotide consists of 20 linked nucleosides and having anucleobase sequence comprising at least 8 contiguous nucleobases of SEQID NO: 77, wherein the modified oligonucleotide comprises: (a) a gapsegment consisting of ten linked deoxynucleosides; (b) a 5′ wing segmentconsisting of five linked nucleosides; (c) a 3′ wing segment consistingof five linked nucleosides; and wherein the gap segment is positionedbetween the 5′ wing segment and the 3′ wing segment, wherein eachnucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar,wherein each internucleoside linkage is a phosphorothioate linkage andwherein each cytosine residue is a 5-methylcytosine.

In certain embodiments, the ANGPTL3 specific inhibitor is a modifiedoligonucleotide with a conjugate group, wherein the modifiedoligonucleotide consists of 20 linked nucleosides and having anucleobase sequence consisting of at least 8 contiguous nucleobases ofSEQ ID NO: 77, wherein the modified oligonucleotide consists of: (a) agap segment consisting often linked deoxynucleosides; (b) a 5′ wingsegment consisting of five linked nucleosides; (c) a 3′ wing segmentconsisting of five linked nucleosides; and wherein the gap segment ispositioned between the 5′ wing segment and the 3′ wing segment, whereineach nucleoside of each wing segment comprises a 2′-O-methoxyethylsugar, wherein each internucleoside linkage is a phosphorothioatelinkage and wherein each cytosine residue is a 5-methylcytosine.

In certain embodiments, the present disclosure provides conjugatedantisense compounds. In certain embodiments, the present disclosureprovides conjugated antisense compounds comprising an antisenseoligonucleotide complementary to a nucleic acid transcript. In certainembodiments, the present disclosure provides methods comprisingcontacting a cell with a conjugated antisense compound comprising anantisense oligonucleotide complementary to a nucleic acid transcript. Incertain embodiments, the present disclosure provides methods comprisingcontacting a cell with a conjugated antisense compound comprising anantisense oligonucleotide and reducing the amount or activity of anucleic acid transcript in a cell.

The asialoglycoprotein receptor (ASGP-R) has been described previously.See e.g., Park et al., PNAS vol. 102, No. 47, pp 17125-17129 (2005).Such receptors are expressed on liver cells, particularly hepatocytes.Further, it has been shown that compounds comprising clusters of threeN-acetylgalactosamine (GalNAc) ligands are capable of binding to theASGP-R, resulting in uptake of the compound into the cell. See e.g.,Khorev et al., Bioorganic and Medicinal Chemistry, 16, 9, pp 5216-5231(May 2008). Accordingly, conjugates comprising such GalNAc clusters havebeen used to facilitate uptake of certain compounds into liver cells,specifically hepatocytes. For example it has been shown that certainGalNAc-containing conjugates increase activity of duplex siRNA compoundsin liver cells in vivo. In such instances, the GalNAc-containingconjugate is typically attached to the sense strand of the siRNA duplex.Since the sense strand is discarded before the antisense strandultimately hybridizes with the target nucleic acid, there is littleconcern that the conjugate will interfere with activity. Typically, theconjugate is attached to the 3′ end of the sense strand of the siRNA.See e.g., U.S. Pat. No. 8,106,022. Certain conjugate groups describedherein are more active and/or easier to synthesize than conjugate groupspreviously described.

In certain embodiments of the present invention, conjugates are attachedto single-stranded antisense compounds, including, but not limited toRNase H based antisense compounds and antisense compounds that altersplicing of a pre-mRNA target nucleic acid. In such embodiments, theconjugate should remain attached to the antisense compound long enoughto provide benefit (improved uptake into cells) but then should eitherbe cleaved, or otherwise not interfere with the subsequent stepsnecessary for activity, such as hybridization to a target nucleic acidand interaction with RNase H or enzymes associated with splicing orsplice modulation. This balance of properties is more important in thesetting of single-stranded antisense compounds than in siRNA compounds,where the conjugate may simply be attached to the sense strand.Disclosed herein are conjugated single-stranded antisense compoundshaving improved potency in liver cells in vivo compared with the sameantisense compound lacking the conjugate. Given the required balance ofproperties for these compounds such improved potency is surprising.

In certain embodiments, conjugate groups herein comprise a cleavablemoiety. As noted, without wishing to be bound by mechanism, it islogical that the conjugate should remain on the compound long enough toprovide enhancement in uptake, but after that, it is desirable for someportion or, ideally, all of the conjugate to be cleaved, releasing theparent compound (e.g., antisense compound) in its most active form. Incertain embodiments, the cleavable moiety is a cleavable nucleoside.Such embodiments take advantage of endogenous nucleases in the cell byattaching the rest of the conjugate (the cluster) to the antisenseoligonucleotide through a nucleoside via one or more cleavable bonds,such as those of a phosphodiester linkage. In certain embodiments, thecluster is bound to the cleavable nucleoside through a phosphodiesterlinkage. In certain embodiments, the cleavable nucleoside is attached tothe antisense oligonucleotide (antisense compound) by a phosphodiesterlinkage. In certain embodiments, the conjugate group may comprise two orthree cleavable nucleosides. In such embodiments, such cleavablenucleosides are linked to one another, to the antisense compound and/orto the cluster via cleavable bonds (such as those of a phosphodiesterlinkage).

Certain conjugates herein do not comprise a cleavable nucleoside andinstead comprise a cleavable bond. It is shown that that sufficientcleavage of the conjugate from the oligonucleotide is provided by atleast one bond that is vulnerable to cleavage in the cell (a cleavablebond).

In certain embodiments, conjugated antisense compounds are prodrugs.Such prodrugs are administered to an animal and are ultimatelymetabolized to a more active form. For example, conjugated antisensecompounds are cleaved to remove all or part of the conjugate resultingin the active (or more active) form of the antisense compound lackingall or some of the conjugate.

In certain embodiments, conjugates are attached at the 5′ end of anoligonucleotide. Certain such 5′-conjugates are cleaved more efficientlythan counterparts having a similar conjugate group attached at the 3′end. In certain embodiments, improved activity may correlate withimproved cleavage. In certain embodiments, oligonucleotides comprising aconjugate at the 5′ end have greater efficacy than oligonucleotidescomprising a conjugate at the 3′ end (see, for example, Examples 56, 81,83, and 84). Further, 5′-attachment allows simpler oligonucleotidesynthesis. Typically, oligonucleotides are synthesized on a solidsupport in the 3′ to 5′ direction. To make a 3′-conjugatedoligonucleotide, typically one attaches a pre-conjugated 3′ nucleosideto the solid support and then builds the oligonucleotide as usual.However, attaching that conjugated nucleoside to the solid support addscomplication to the synthesis. Further, using that approach, theconjugate is then present throughout the synthesis of theoligonucleotide and can become degraded during subsequent steps or maylimit the sorts of reactions and reagents that can be used. Using thestructures and techniques described herein for 5′-conjugatedoligonucleotides, one can synthesize the oligonucleotide using standardautomated techniques and introduce the conjugate with the final(5′-most) nucleoside or after the oligonucleotide has been cleaved fromthe solid support.

In view of the art and the present disclosure, one of ordinary skill caneasily make any of the conjugates and conjugated oligonucleotidesherein. Moreover, synthesis of certain such conjugates and conjugatedoligonucleotides disclosed herein is easier and/or requires few steps,and is therefore less expensive than that of conjugates previouslydisclosed, providing advantages in manufacturing. For example, thesynthesis of certain conjugate groups consists of fewer synthetic steps,resulting in increased yield, relative to conjugate groups previouslydescribed. Conjugate groups such as GalNAc₃-10 in Example 46 andGalNAc₃-7 in Example 48 are much simpler than previously describedconjugates such as those described in U.S. Pat. No. 8,106,022 or U.S.Pat. No. 7,262,177 that require assembly of more chemical intermediates.Accordingly, these and other conjugates described herein have advantagesover previously described compounds for use with any oligonucleotide,including single-stranded oligonucleotides and either strand ofdouble-stranded oligonucleotides (e.g., siRNA).

Similarly, disclosed herein are conjugate groups having only one or twoGalNAc ligands. As shown, such conjugates groups improve activity ofantisense compounds. Such compounds are much easier to prepare thanconjugates comprising three GalNAc ligands. Conjugate groups comprisingone or two GalNAc ligands may be attached to any antisense compounds,including single-stranded oligonucleotides and either strand ofdouble-stranded oligonucleotides (e.g., siRNA).

In certain embodiments, the conjugates herein do not substantially altercertain measures of tolerability. For example, it is shown herein thatconjugated antisense compounds are not more immunogenic thanunconjugated parent compounds. Since potency is improved, embodiments inwhich tolerability remains the same (or indeed even if tolerabilityworsens only slightly compared to the gains in potency) have improvedproperties for therapy.

In certain embodiments, conjugation allows one to alter antisensecompounds in ways that have less attractive consequences in the absenceof conjugation. For example, in certain embodiments, replacing one ormore phosphorothioate linkages of a fully phosphorothioate antisensecompound with phosphodiester linkages results in improvement in somemeasures of tolerability. For example, in certain instances, suchantisense compounds having one or more phosphodiester are lessimmunogenic than the same compound in which each linkage is aphosphorothioate. However, in certain instances, as shown in Example 26,that same replacement of one or more phosphorothioate linkages withphosphodiester linkages also results in reduced cellular uptake and/orloss in potency. In certain embodiments, conjugated antisense compoundsdescribed herein tolerate such change in linkages with little or no lossin uptake and potency when compared to the conjugatedfull-phosphorothioate counterpart. In fact, in certain embodiments, forexample, in Examples 44, 57, 59, and 86, oligonucleotides comprising aconjugate and at least one phosphodiester internucleoside linkageactually exhibit increased potency in vivo even relative to a fullphosphorothioate counterpart also comprising the same conjugate.Moreover, since conjugation results in substantial increases inuptake/potency a small loss in that substantial gain may be acceptableto achieve improved tolerability. Accordingly, in certain embodiments,conjugated antisense compounds comprise at least one phosphodiesterlinkage.

In certain embodiments, conjugation of antisense compounds hereinresults in increased delivery, uptake and activity in hepatocytes. Thus,more compound is delivered to liver tissue. However, in certainembodiments, that increased delivery alone does not explain the entireincrease in activity. In certain such embodiments, more compound entershepatocytes. In certain embodiments, even that increased hepatocyteuptake does not explain the entire increase in activity. In suchembodiments, productive uptake of the conjugated compound is increased.For example, as shown in Example 102, certain embodiments ofGalNAc-containing conjugates increase enrichment of antisenseoligonucleotides in hepatocytes versus non-parenchymal cells. Thisenrichment is beneficial for oligonucleotides that target genes that areexpressed in hepatocytes.

In certain embodiments, conjugated antisense compounds herein result inreduced kidney exposure. For example, as shown in Example 20, theconcentrations of antisense oligonucleotides comprising certainembodiments of GalNAc-containing conjugates are lower in the kidney thanthat of antisense oligonucleotides lacking a GalNAc-containingconjugate. This has several beneficial therapeutic implications. Fortherapeutic indications where activity in the kidney is not sought,exposure to kidney risks kidney toxicity without corresponding benefit.Moreover, high concentration in kidney typically results in loss ofcompound to the urine resulting in faster clearance. Accordingly fornon-kidney targets, kidney accumulation is undesired.

In certain embodiments, the present disclosure provides conjugatedantisense compounds represented by the formula:

A-B—C-D E-F)_(q)

wherein

A is the antisense oligonucleotide;

B is the cleavable moiety

C is the conjugate linker

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In the above diagram and in similar diagrams herein, the branching group“D” branches as many times as is necessary to accommodate the number of(E-F) groups as indicated by “q”. Thus, where q=1, the formula is:

A-B—C-D-E-F

where q=2, the formula is:

where q=3, the formula is:

where q=4, the formula is:

where q=5, the formula is:

In certain embodiments, conjugated antisense compounds are providedhaving the structure:

In certain embodiments, conjugated antisense compounds are providedhaving the structure:

In certain embodiments, conjugated antisense compounds are providedhaving the structure:

In certain embodiments, conjugated antisense compounds are providedhaving the structure:

In embodiments having more than one of a particular variable (e.g., morethan one “m” or “n”), unless otherwise indicated, each such particularvariable is selected independently. Thus, for a structure having morethan one n, each n is selected independently, so they may or may not bethe same as one another.

In certain embodiments, the present disclosure provides conjugatedantisense compounds represented by the following structure. In certainembodiments, the antisense compound comprises the modifiedoligonucleotide ISIS 563580 with a 5′-X, wherein X is a conjugate groupcomprising GalNAc. In certain embodiments, the antisense compoundconsists of the modified oligonucleotide ISIS 563580 with a 5′-X,wherein X is a conjugate group comprising GalNAc.

In certain embodiments, the present disclosure provides conjugatedantisense compounds represented by the following structure. In certainembodiments, the antisense compound comprises the conjugated modifiedoligonucleotide ISIS 703801. In certain embodiments, the antisensecompound consists of the conjugated modified oligonucleotide ISIS703801.

In certain embodiments, the present disclosure provides conjugatedantisense compounds represented by the following structure. In certainembodiments, the antisense compound comprises the conjugated modifiedoligonucleotide ISIS 703802. In certain embodiments, the antisensecompound consists of the conjugated modified oligonucleotide ISIS703802.

In certain embodiments, the present disclosure provides conjugatedantisense compounds represented by the following structure. In certainembodiments, the antisense compound comprises a modified oligonucleotidewith the nucleobase sequence of SEQ ID NO: 77 with a 5′-GalNAc withvariability in the sugar mods of the wings. In certain embodiments, theantisense compound consists of a modified oligonucleotide with thenucleobase sequence of SEQ ID NO: 77 with a 5′-GalNAc with variabilityin the sugar mods of the wings.

wherein either R′ is —OCH₂CH₂OCH₃ (MOE) and R² is H; or R¹ and R²together form a bridge, wherein R¹ is —O— and R² is —CH₂—, —CH(CH₃)—, or—CH₂CH₂—, and IV and R² are directly connected such that the resultingbridge is selected from: —O—CH₂—, —O—CH(CH₃)—, and —O—CH₂CH₂—;

and for each pair of R³ and R⁴ on the same ring, independently for eachring: either R³ is selected from H and —OCH₂CH₂OCH₃ and R⁴ is H; or R³and R⁴ together form a bridge, wherein R³ is —O—, and R⁴ is —CH₂—,—CH(CH₃)—, or —CH₂CH₂— and R³ and R⁴ are directly connected such thatthe resulting bridge is selected from: —O—CH₂—, —O—CH(CH₃)—, and—O—CH₂CH₂—;

and R⁵ is selected from H and —CH₃;

and Z is selected from S⁻ and O⁻.

Certain embodiments provide a composition comprising a conjugatedantisense compound described herein, or a salt thereof, and apharmaceutically acceptable carrier or diluent.

In certain embodiments, the modulation of ANGPTL3 expression occurs in acell or tissue. In certain embodiments, the modulations occur in a cellor tissue in an animal. In certain embodiments, the animal is a human.In certain embodiments, the modulation is a reduction in ANGPTL3 mRNAlevel. In certain embodiments, the modulation is a reduction in ANGPTL3protein level. In certain embodiments, both ANGPTL3 mRNA and proteinlevels are reduced. Such reduction may occur in a time-dependent or in adose-dependent manner.

Certain embodiments provide compositions and methods for use in therapy.Certain embodiments provide compositions and methods for preventing,treating, delaying, slowing the progression and/or ameliorating ANGPTL3related diseases, disorders, and conditions. In certain embodiments,such diseases, disorders, and conditions are cardiovascular and/ormetabolic diseases, disorders, and conditions. In certain embodiments,the compositions and methods for therapy include administering anANGPTL3 specific inhibitor to an individual in need thereof. In certainembodiments, the ANGPTL3 specific inhibitor is a nucleic acid. Incertain embodiments, the nucleic acid is an antisense compound. Incertain embodiments, the antisense compound is a modifiedoligonucleotide. In certain embodiments, the antisense compound is amodified oligonucleotide with a conjugate group attached.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. Herein, the use ofthe singular includes the plural unless specifically stated otherwise.As used herein, the use of “or” means “and/or” unless stated otherwise.Furthermore, the use of the term “including” as well as other forms,such as “includes” and “included”, is not limiting. Also, terms such as“element” or “component” encompass both elements and componentscomprising one unit and elements and components that comprise more thanone subunit, unless specifically stated otherwise.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including, but not limited to, patents, patent applications, articles,books, and treatises, are hereby expressly incorporated-by-reference forthe portions of the document discussed herein, as well as in theirentirety.

Definitions

Unless specific definitions are provided, the nomenclature utilized inconnection with, and the procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques can be used for chemical synthesis, andchemical analysis. Certain such techniques and procedures may be foundfor example in “Carbohydrate Modifications in Antisense Research” Editedby Sangvi and Cook, American Chemical Society, Washington D.C., 1994;“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,21^(st) edition, 2005; and “Antisense Drug Technology, Principles,Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press,Boca Raton, Fla.; and Sambrook et al., “Molecular Cloning, A laboratoryManual,” 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, whichare hereby incorporated by reference for any purpose. Where permitted,all patents, applications, published applications and otherpublications, GENBANK Accession Numbers and associated sequenceinformation obtainable through databases such as National Center forBiotechnology Information (NCBI) and other data referred to throughoutin the disclosure herein are incorporated by reference for the portionsof the document discussed herein, as well as in their entirety.

Unless otherwise indicated, the following terms have the followingmeanings:

As used herein, “nucleoside” means a compound comprising a nucleobasemoiety and a sugar moiety. Nucleosides include, but are not limited to,naturally occurring nucleosides (as found in DNA and RNA) and modifiednucleosides. Nucleosides may be linked to a phosphate moiety.

As used herein, “chemical modification” means a chemical difference in acompound when compared to a naturally occurring counterpart. Chemicalmodifications of oligonucleotides include nucleoside modifications(including sugar moiety modifications and nucleobase modifications) andinternucleoside linkage modifications. In reference to anoligonucleotide, chemical modification does not include differences onlyin nucleobase sequence.

As used herein, “furanosyl” means a structure comprising a 5-memberedring comprising four carbon atoms and one oxygen atom.

As used herein, “naturally occurring sugar moiety” means a ribofuranosylas found in naturally occurring RNA or a deoxyribofuranosyl as found innaturally occurring DNA.

As used herein, “sugar moiety” means a naturally occurring sugar moietyor a modified sugar moiety of a nucleoside.

As used herein, “modified sugar moiety” means a substituted sugar moietyor a sugar surrogate.

As used herein, “substituted sugar moiety” means a furanosyl that is nota naturally occurring sugar moiety. Substituted sugar moieties include,but are not limited to furanosyls comprising substituents at the2′-position, the 3′-position, the 5′-position and/or the 4′-position.Certain substituted sugar moieties are bicyclic sugar moieties.

As used herein, “2′-substituted sugar moiety” means a furanosylcomprising a substituent at the 2′-position other than H or OH. Unlessotherwise indicated, a 2′-substituted sugar moiety is not a bicyclicsugar moiety (i.e., the 2′-substituent of a 2′-substituted sugar moietydoes not form a bridge to another atom of the furanosyl ring.

As used herein, “MOE” means —OCH₂CH₂OCH₃.

As used herein, “2′-F nucleoside” refers to a nucleoside comprising asugar comprising fluorine at the 2′ position. Unless otherwiseindicated, the fluorine in a 2′-F nucleoside is in the ribo position(replacing the OH of a natural ribose).

As used herein the term “sugar surrogate” means a structure that doesnot comprise a furanosyl and that is capable of replacing the naturallyoccurring sugar moiety of a nucleoside, such that the resultingnucleoside sub-units are capable of linking together and/or linking toother nucleosides to form an oligomeric compound which is capable ofhybridizing to a complementary oligomeric compound. Such structuresinclude rings comprising a different number of atoms than furanosyl(e.g., 4, 6, or 7-membered rings); replacement of the oxygen of afuranosyl with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen); orboth a change in the number of atoms and a replacement of the oxygen.Such structures may also comprise substitutions corresponding to thosedescribed for substituted sugar moieties (e.g., 6-membered carbocyclicbicyclic sugar surrogates optionally comprising additionalsubstituents). Sugar surrogates also include more complex sugarreplacements (e.g., the non-ring systems of peptide nucleic acid). Sugarsurrogates include without limitation morpholinos, cyclohexenyls andcyclohexitols.

As used herein, “bicyclic sugar moiety” means a modified sugar moietycomprising a 4 to 7 membered ring (including but not limited to afuranosyl) comprising a bridge connecting two atoms of the 4 to 7membered ring to form a second ring, resulting in a bicyclic structure.In certain embodiments, the 4 to 7 membered ring is a sugar ring. Incertain embodiments the 4 to 7 membered ring is a furanosyl. In certainsuch embodiments, the bridge connects the 2′-carbon and the 4′-carbon ofthe furanosyl.

As used herein, “nucleotide” means a nucleoside further comprising aphosphate linking group. As used herein, “linked nucleosides” may or maynot be linked by phosphate linkages and thus includes, but is notlimited to “linked nucleotides.” As used herein, “linked nucleosides”are nucleosides that are connected in a continuous sequence (i.e. noadditional nucleosides are present between those that are linked).

As used herein, “nucleobase” means a group of atoms that can be linkedto a sugar moiety to create a nucleoside that is capable ofincorporation into an oligonucleotide, and wherein the group of atoms iscapable of bonding with a complementary naturally occurring nucleobaseof another oligonucleotide or nucleic acid. Nucleobases may be naturallyoccurring or may be modified. “Nucleobase sequence” means the order ofcontiguous nucleobases independent of any sugar, linkage, or nucleobasemodification.

As used herein the terms, “unmodified nucleobase” or “naturallyoccurring nucleobase” means the naturally occurring heterocyclicnucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G),and the pyrimidine bases thymine (T), cytosine (C) (including 5-methylC), and uracil (U).

As used herein, “modified nucleobase” means any nucleobase that is not anaturally occurring nucleobase.

As used herein, “modified nucleoside” means a nucleoside comprising atleast one chemical modification compared to naturally occurring RNA orDNA nucleosides. Modified nucleosides comprise a modified sugar moietyand/or a modified nucleobase.

As used herein, “bicyclic nucleoside” or “BNA” means a nucleosidecomprising a bicyclic sugar moiety.

As used herein, “constrained ethyl nucleoside” or “cEt” means anucleoside comprising a bicyclic sugar moiety comprising a4′-CH(CH₃)—O-2′ bridge.

As used herein, “locked nucleic acid nucleoside” or “LNA” means anucleoside comprising a bicyclic sugar moiety comprising a 4′-CH₂—O-2′bridge.

As used herein, “2′-substituted nucleoside” means a nucleosidecomprising a substituent at the 2′-position other than H or OH. Unlessotherwise indicated, a 2′-substituted nucleoside is not a bicyclicnucleoside.

As used herein, “deoxynucleoside” means a nucleoside comprising 2′-Hfuranosyl sugar moiety, as found in naturally occurringdeoxyribonucleosides (DNA). In certain embodiments, a 2′-deoxynucleosidemay comprise a modified nucleobase or may comprise an RNA nucleobase(e.g., uracil).

As used herein, “oligonucleotide” means a compound comprising aplurality of linked nucleosides. In certain embodiments, anoligonucleotide comprises one or more unmodified ribonucleosides (RNA)and/or unmodified deoxyribonucleosides (DNA) and/or one or more modifiednucleosides.

As used herein “oligonucleoside” means an oligonucleotide in which noneof the internucleoside linkages contains a phosphorus atom. As usedherein, oligonucleotides include oligonucleosides.

As used herein, “modified oligonucleotide” means an oligonucleotidecomprising at least one modified nucleoside and/or at least one modifiedinternucleoside linkage.

As used herein, “linkage” or “linking group” means a group of atoms thatlink together two or more other groups of atoms.

As used herein “internucleoside linkage” means a covalent linkagebetween adjacent nucleosides in an oligonucleotide.

As used herein “naturally occurring internucleoside linkage” means a 3′to 5′ phosphodiester linkage.

As used herein, “modified internucleoside linkage” means anyinternucleoside linkage other than a naturally occurring internucleosidelinkage.

As used herein, “terminal internucleoside linkage” means the linkagebetween the last two nucleosides of an oligonucleotide or defined regionthereof.

As used herein, “phosphorus linking group” means a linking groupcomprising a phosphorus atom.

Phosphorus linking groups include without limitation groups having theformula:

wherein:

R_(a) and R are each, independently, O, S, CH₂, NH, or NJ₁ wherein J₁ isC₁-C₆ alkyl or substituted C₁-C₆ alkyl;

R_(b) is O or S;

R_(c) is OH, SH, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy,substituted C₁-C₆ alkoxy, amino or substituted amino; and

J₁ is R_(b) is O or S.

Phosphorus linking groups include without limitation, phosphodiester,phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate,phosphorothioamidate, thionoalkylphosphonate, phosphotriesters,thionoalkylphosphotriester and boranophosphate.

As used herein, “internucleoside phosphorus linking group” means aphosphorus linking group that directly links two nucleosides.

As used herein, “non-internucleoside phosphorus linking group” means aphosphorus linking group that does not directly link two nucleosides. Incertain embodiments, a non-internucleoside phosphorus linking grouplinks a nucleoside to a group other than a nucleoside. In certainembodiments, a non-internucleoside phosphorus linking group links twogroups, neither of which is a nucleoside.

As used herein, “neutral linking group” means a linking group that isnot charged. Neutral linking groups include without limitationphosphotriesters, methylphosphonates, MMI (—CH₂—N(CH₃)—O—), amide-3(—CH₂—C(═O)—N(H)—), amide-4 (—CH₂—N(H)—C(═O)—), formacetal (—O—CH₂—O—),and thioformacetal (—S—CH₂—O—).

Further neutral linking groups include nonionic linkages comprisingsiloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide,sulfonate ester and amides (See for example: Carbohydrate Modificationsin Antisense Research; Y. S. Sanghvi and P. D. Cook Eds. ACS SymposiumSeries 580; Chapters 3 and 4, (pp. 40-65)). Further neutral linkinggroups include nonionic linkages comprising mixed N, O, S and CH₂component parts.

As used herein, “internucleoside neutral linking group” means a neutrallinking group that directly links two nucleosides.

As used herein, “non-internucleoside neutral linking group” means aneutral linking group that does not directly link two nucleosides. Incertain embodiments, a non-internucleoside neutral linking group links anucleoside to a group other than a nucleoside. In certain embodiments, anon-internucleoside neutral linking group links two groups, neither ofwhich is a nucleoside.

As used herein, “oligomeric compound” means a polymeric structurecomprising two or more sub-structures. In certain embodiments, anoligomeric compound comprises an oligonucleotide. In certainembodiments, an oligomeric compound comprises one or more conjugategroups and/or terminal groups. In certain embodiments, an oligomericcompound consists of an oligonucleotide. Oligomeric compounds alsoinclude naturally occurring nucleic acids. In certain embodiments, anoligomeric compound comprises a backbone of one or more linked monomericsubunits where each linked monomeric subunit is directly or indirectlyattached to a heterocyclic base moiety. In certain embodiments,oligomeric compounds may also include monomeric subunits that are notlinked to a heterocyclic base moiety, thereby providing abasic sites. Incertain embodiments, the linkages joining the monomeric subunits, thesugar moieties or surrogates and the heterocyclic base moieties can beindependently modified. In certain embodiments, the linkage-sugar unit,which may or may not include a heterocyclic base, may be substitutedwith a mimetic such as the monomers in peptide nucleic acids.

As used herein, “terminal group” means one or more atom attached toeither, or both, the 3′ end or the 5′ end of an oligonucleotide. Incertain embodiments a terminal group is a conjugate group. In certainembodiments, a terminal group comprises one or more terminal groupnucleosides.

As used herein, “conjugate” or “conjugate group” means an atom or groupof atoms bound to an oligonucleotide or oligomeric compound. In general,conjugate groups modify one or more properties of the compound to whichthey are attached, including, but not limited to pharmacodynamic,pharmacokinetic, binding, absorption, cellular distribution, cellularuptake, charge and/or clearance properties.

As used herein, “conjugate linker” or “linker” in the context of aconjugate group means a portion of a conjugate group comprising any atomor group of atoms and which covalently link (1) an oligonucleotide toanother portion of the conjugate group or (2) two or more portions ofthe conjugate group.

Conjugate groups are shown herein as radicals, providing a bond forforming covalent attachment to an oligomeric compound such as anantisense oligonucleotide. In certain embodiments, the point ofattachment on the oligomeric compound is the 3′-oxygen atom of the3′-hydroxyl group of the 3′ terminal nucleoside of the oligomericcompound. In certain embodiments the point of attachment on theoligomeric compound is the 5′-oxygen atom of the 5′-hydroxyl group ofthe 5′ terminal nucleoside of the oligomeric compound. In certainembodiments, the bond for forming attachment to the oligomeric compoundis a cleavable bond. In certain such embodiments, such cleavable bondconstitutes all or part of a cleavable moiety.

In certain embodiments, conjugate groups comprise a cleavable moiety(e.g., a cleavable bond or cleavable nucleoside) and a carbohydratecluster portion, such as a GalNAc cluster portion. Such carbohydratecluster portion comprises: a targeting moiety and, optionally, aconjugate linker. In certain embodiments, the carbohydrate clusterportion is identified by the number and identity of the ligand. Forexample, in certain embodiments, the carbohydrate cluster portioncomprises 3 GalNAc groups and is designated “GalNAc₃”. In certainembodiments, the carbohydrate cluster portion comprises 4 GalNAc groupsand is designated “GalNAc₄”. Specific carbohydrate cluster portions(having specific tether, branching and conjugate linker groups) aredescribed herein and designated by Roman numeral followed by subscript“a”. Accordingly “GalNac3-1_(a)” refers to a specific carbohydratecluster portion of a conjugate group having 3 GalNac groups andspecifically identified tether, branching and linking groups. Suchcarbohydrate cluster fragment is attached to an oligomeric compound viaa cleavable moiety, such as a cleavable bond or cleavable nucleoside.

As used herein, “cleavable moiety” means a bond or group that is capableof being split under physiological conditions. In certain embodiments, acleavable moiety is cleaved inside a cell or sub-cellular compartments,such as a lysosome. In certain embodiments, a cleavable moiety iscleaved by endogenous enzymes, such as nucleases. In certainembodiments, a cleavable moiety comprises a group of atoms having one,two, three, four, or more than four cleavable bonds.

As used herein, “cleavable bond” means any chemical bond capable ofbeing split. In certain embodiments, a cleavable bond is selected fromamong: an amide, a polyamide, an ester, an ether, one or both esters ofa phosphodiester, a phosphate ester, a carbamate, a di-sulfide, or apeptide.

As used herein, “carbohydrate cluster” means a compound having one ormore carbohydrate residues attached to a scaffold or linker group. (see,e.g., Maier et al., “Synthesis of Antisense Oligonucleotides Conjugatedto a Multivalent Carbohydrate Cluster for Cellular Targeting,”Bioconjugate Chemistry, 2003, (14): 18-29, which is incorporated hereinby reference in its entirety, or Rensen et al., “Design and Synthesis ofNovel N-Acetylgalactosamine-Terminated Glycolipids for Targeting ofLipoproteins to the Hepatic Asiaglycoprotein Receptor,” J. Med. Chem.2004, (47): 5798-5808, for examples of carbohydrate conjugate clusters).

As used herein, “modified carbohydrate” means any carbohydrate havingone or more chemical modifications relative to naturally occurringcarbohydrates.

As used herein, “carbohydrate derivative” means any compound which maybe synthesized using a carbohydrate as a starting material orintermediate.

As used herein, “carbohydrate” means a naturally occurring carbohydrate,a modified carbohydrate, or a carbohydrate derivative.

As used herein “protecting group” means any compound or protecting groupknown to those having skill in the art. Non-limiting examples ofprotecting groups may be found in “Protective Groups in OrganicChemistry”, T. W. Greene, P. G. M. Wuts, ISBN 0-471-62301-6, John Wiley& Sons, Inc, New York, which is incorporated herein by reference in itsentirety.

As used herein, “single-stranded” means an oligomeric compound that isnot hybridized to its complement and which lacks sufficientself-complementarity to form a stable self-duplex.

As used herein, “double stranded” means a pair of oligomeric compoundsthat are hybridized to one another or a single self-complementaryoligomeric compound that forms a hairpin structure. In certainembodiments, a double-stranded oligomeric compound comprises a first anda second oligomeric compound.

As used herein, “antisense compound” means a compound comprising orconsisting of an oligonucleotide at least a portion of which iscomplementary to a target nucleic acid to which it is capable ofhybridizing, resulting in at least one antisense activity.

As used herein, “antisense activity” means any detectable and/ormeasurable change attributable to the hybridization of an antisensecompound to its target nucleic acid. In certain embodiments, antisenseactivity includes modulation of the amount or activity of a targetnucleic acid transcript (e.g. mRNA). In certain embodiments, antisenseactivity includes modulation of the splicing of pre-mRNA.

As used herein, “RNase H based antisense compound” means an antisensecompound wherein at least some of the antisense activity of theantisense compound is attributable to hybridization of the antisensecompound to a target nucleic acid and subsequent cleavage of the targetnucleic acid by RNase H.

As used herein, “RISC based antisense compound” means an antisensecompound wherein at least some of the antisense activity of theantisense compound is attributable to the RNA Induced Silencing Complex(RISC).

As used herein, “detecting” or “measuring” means that a test or assayfor detecting or measuring is performed. Such detection and/or measuringmay result in a value of zero. Thus, if a test for detection ormeasuring results in a finding of no activity (activity of zero), thestep of detecting or measuring the activity has nevertheless beenperformed.

As used herein, “detectable and/or measureable activity” means astatistically significant activity that is not zero.

As used herein, “essentially unchanged” means little or no change in aparticular parameter, particularly relative to another parameter whichchanges much more. In certain embodiments, a parameter is essentiallyunchanged when it changes less than 5%. In certain embodiments, aparameter is essentially unchanged if it changes less than two-foldwhile another parameter changes at least ten-fold. For example, incertain embodiments, an antisense activity is a change in the amount ofa target nucleic acid. In certain such embodiments, the amount of anon-target nucleic acid is essentially unchanged if it changes much lessthan the target nucleic acid does, but the change need not be zero.

As used herein, “expression” means the process by which a geneultimately results in a protein.

Expression includes, but is not limited to, transcription,post-transcriptional modification (e.g., splicing, polyadenlyation,addition of 5′-cap), and translation.

As used herein, “target nucleic acid” means a nucleic acid molecule towhich an antisense compound is intended to hybridize to result in adesired antisense activity. Antisense oligonucleotides have sufficientcomplementarity to their target nucleic acids to allow hybridizationunder physiological conditions.

As used herein, “nucleobase complementarity” or “complementarity” whenin reference to nucleobases means a nucleobase that is capable of basepairing with another nucleobase. For example, in DNA, adenine (A) iscomplementary to thymine (T). For example, in RNA, adenine (A) iscomplementary to uracil (U). In certain embodiments, complementarynucleobase means a nucleobase of an antisense compound that is capableof base pairing with a nucleobase of its target nucleic acid. Forexample, if a nucleobase at a certain position of an antisense compoundis capable of hydrogen bonding with a nucleobase at a certain positionof a target nucleic acid, then the position of hydrogen bonding betweenthe oligonucleotide and the target nucleic acid is considered to becomplementary at that nucleobase pair. Nucleobases comprising certainmodifications may maintain the ability to pair with a counterpartnucleobase and thus, are still capable of nucleobase complementarity.

As used herein, “non-complementary” in reference to nucleobases means apair of nucleobases that do not form hydrogen bonds with one another.

As used herein, “complementary” in reference to oligomeric compounds(e.g., linked nucleosides, oligonucleotides, or nucleic acids) means thecapacity of such oligomeric compounds or regions thereof to hybridize toanother oligomeric compound or region thereof through nucleobasecomplementarity. Complementary oligomeric compounds need not havenucleobase complementarity at each nucleoside. Rather, some mismatchesare tolerated. In certain embodiments, complementary oligomericcompounds or regions are complementary at 70% of the nucleobases (70%complementary). In certain embodiments, complementary oligomericcompounds or regions are 80% complementary. In certain embodiments,complementary oligomeric compounds or regions are 90% complementary. Incertain embodiments, complementary oligomeric compounds or regions are95% complementary. In certain embodiments, complementary oligomericcompounds or regions are 100% complementary.

As used herein, “mismatch” means a nucleobase of a first oligomericcompound that is not capable of pairing with a nucleobase at acorresponding position of a second oligomeric compound, when the firstand second oligomeric compound are aligned. Either or both of the firstand second oligomeric compounds may be oligonucleotides.

As used herein, “hybridization” means the pairing of complementaryoligomeric compounds (e.g., an antisense compound and its target nucleicacid). While not limited to a particular mechanism, the most commonmechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleobases.

As used herein, “specifically hybridizes” means the ability of anoligomeric compound to hybridize to one nucleic acid site with greateraffinity than it hybridizes to another nucleic acid site.

As used herein, “fully complementary” in reference to an oligonucleotideor portion thereof means that each nucleobase of the oligonucleotide orportion thereof is capable of pairing with a nucleobase of acomplementary nucleic acid or contiguous portion thereof. Thus, a fullycomplementary region comprises no mismatches or unhybridized nucleobasesin either strand.

As used herein, “percent complementarity” means the percentage ofnucleobases of an oligomeric compound that are complementary to anequal-length portion of a target nucleic acid. Percent complementarityis calculated by dividing the number of nucleobases of the oligomericcompound that are complementary to nucleobases at correspondingpositions in the target nucleic acid by the total length of theoligomeric compound.

As used herein, “percent identity” means the number of nucleobases in afirst nucleic acid that are the same type (independent of chemicalmodification) as nucleobases at corresponding positions in a secondnucleic acid, divided by the total number of nucleobases in the firstnucleic acid.

As used herein, “modulation” means a change of amount or quality of amolecule, function, or activity when compared to the amount or qualityof a molecule, function, or activity prior to modulation. For example,modulation includes the change, either an increase (stimulation orinduction) or a decrease (inhibition or reduction) in gene expression.As a further example, modulation of expression can include a change insplice site selection of pre-mRNA processing, resulting in a change inthe absolute or relative amount of a particular splice-variant comparedto the amount in the absence of modulation.

As used herein, “chemical motif” means a pattern of chemicalmodifications in an oligonucleotide or a region thereof. Motifs may bedefined by modifications at certain nucleosides and/or at certainlinking groups of an oligonucleotide.

As used herein, “nucleoside motif” means a pattern of nucleosidemodifications in an oligonucleotide or a region thereof. The linkages ofsuch an oligonucleotide may be modified or unmodified. Unless otherwiseindicated, motifs herein describing only nucleosides are intended to benucleoside motifs. Thus, in such instances, the linkages are notlimited.

As used herein, “sugar motif” means a pattern of sugar modifications inan oligonucleotide or a region thereof.

As used herein, “linkage motif” means a pattern of linkage modificationsin an oligonucleotide or region thereof. The nucleosides of such anoligonucleotide may be modified or unmodified. Unless otherwiseindicated, motifs herein describing only linkages are intended to belinkage motifs. Thus, in such instances, the nucleosides are notlimited.

As used herein, “nucleobase modification motif” means a pattern ofmodifications to nucleobases along an oligonucleotide. Unless otherwiseindicated, a nucleobase modification motif is independent of thenucleobase sequence.

As used herein, “sequence motif” means a pattern of nucleobases arrangedalong an oligonucleotide or portion thereof. Unless otherwise indicated,a sequence motif is independent of chemical modifications and thus mayhave any combination of chemical modifications, including no chemicalmodifications.

As used herein, “type of modification” in reference to a nucleoside or anucleoside of a “type” means the chemical modification of a nucleosideand includes modified and unmodified nucleosides. Accordingly, unlessotherwise indicated, a “nucleoside having a modification of a firsttype” may be an unmodified nucleoside.

As used herein, “differently modified” mean chemical modifications orchemical substituents that are different from one another, includingabsence of modifications. Thus, for example, a MOE nucleoside and anunmodified DNA nucleoside are “differently modified,” even though theDNA nucleoside is unmodified. Likewise, DNA and RNA are “differentlymodified,” even though both are naturally-occurring unmodifiednucleosides. Nucleosides that are the same but for comprising differentnucleobases are not differently modified. For example, a nucleosidecomprising a 2′-OMe modified sugar and an unmodified adenine nucleobaseand a nucleoside comprising a 2′-OMe modified sugar and an unmodifiedthymine nucleobase are not differently modified.

As used herein, “the same type of modifications” refers to modificationsthat are the same as one another, including absence of modifications.Thus, for example, two unmodified DNA nucleosides have “the same type ofmodification,” even though the DNA nucleoside is unmodified. Suchnucleosides having the same type modification may comprise differentnucleobases.

As used herein, “separate regions” means portions of an oligonucleotidewherein the chemical modifications or the motif of chemicalmodifications of any neighboring portions include at least onedifference to allow the separate regions to be distinguished from oneanother.

As used herein, “pharmaceutically acceptable carrier or diluent” meansany substance suitable for use in administering to an animal. In certainembodiments, a pharmaceutically acceptable carrier or diluent is sterilesaline. In certain embodiments, such sterile saline is pharmaceuticalgrade saline.

As used herein the term “metabolic disorder” means a disease orcondition principally characterized by dysregulation of metabolism—thecomplex set of chemical reactions associated with breakdown of food toproduce energy.

As used herein, the term “Cardiovascular disease” or “cardiovasculardisorder” means a disease or condition principally characterized byimpaired function of the heart or blood vessels. Examples ofcardiovascular diseases or disorders include, but are not limited to,aneurysm, angina, arrhythmia, atherosclerosis, cerebrovascular disease(stroke), coronary heart disease, hypertension, dyslipidemia,hyperlipidemia, and hypercholesterolemia.

As used herein the term “mono or polycyclic ring system” is meant toinclude all ring systems selected from single or polycyclic radical ringsystems wherein the rings are fused or linked and is meant to beinclusive of single and mixed ring systems individually selected fromaliphatic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl,heterocyclic, heteroaryl, heteroaromatic and heteroarylalkyl. Such monoand poly cyclic structures can contain rings that each have the samelevel of saturation or each, independently, have varying degrees ofsaturation including fully saturated, partially saturated or fullyunsaturated. Each ring can comprise ring atoms selected from C, N, O andS to give rise to heterocyclic rings as well as rings comprising only Cring atoms which can be present in a mixed motif such as for examplebenzimidazole wherein one ring has only carbon ring atoms and the fusedring has two nitrogen atoms. The mono or polycyclic ring system can befurther substituted with substituent groups such as for examplephthalimide which has two ═O groups attached to one of the rings. Monoor polycyclic ring systems can be attached to parent molecules usingvarious strategies such as directly through a ring atom, fused throughmultiple ring atoms, through a substituent group or through abifunctional linking moiety.

As used herein, “prodrug” means an inactive or less active form of acompound which, when administered to a subject, is metabolized to formthe active, or more active, compound (e.g., drug).

As used herein, “substituent” and “substituent group,” means an atom orgroup that replaces the atom or group of a named parent compound. Forexample a substituent of a modified nucleoside is any atom or group thatdiffers from the atom or group found in a naturally occurring nucleoside(e.g., a modified 2′-substuent is any atom or group at the 2′-positionof a nucleoside other than H or OH). Substituent groups can be protectedor unprotected. In certain embodiments, compounds of the presentdisclosure have substituents at one or at more than one position of theparent compound. Substituents may also be further substituted with othersubstituent groups and may be attached directly or via a linking groupsuch as an alkyl or hydrocarbyl group to a parent compound.

Likewise, as used herein, “substituent” in reference to a chemicalfunctional group means an atom or group of atoms that differs from theatom or a group of atoms normally present in the named functional group.

In certain embodiments, a substituent replaces a hydrogen atom of thefunctional group (e.g., in certain embodiments, the substituent of asubstituted methyl group is an atom or group other than hydrogen whichreplaces one of the hydrogen atoms of an unsubstituted methyl group).Unless otherwise indicated, groups amenable for use as substituentsinclude without limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl,acyl (—C(O)R_(aa)), carboxyl (—C(O)O—R_(aa)), aliphatic groups,alicyclic groups, alkoxy, substituted oxy (—O—R_(aa)), aryl, aralkyl,heterocyclic radical, heteroaryl, heteroarylalkyl, amino(—N(R_(bb))(R_(cc))), imino(=NR_(bb)), amido (—C(O)N—(R_(bb))(R_(cc)) or—N(R_(bb))C(O)R_(aa)), azido (—N₃), nitro (—NO₂), cyano (—CN), carbamido(—OC(O)N(R_(bb))(R_(cc)) or —N(R_(bb))C(O)OR_(aa)), ureido(—N(R_(bb))C(O)N(R_(bb))(R_(cc))), thioureido(—N(R_(bb))C(S)N(R_(bb))(R_(cc))), guanidinyl(—N(R_(bb))C(═NR_(bb))N(R_(bb))(R_(cc))), amidinyl(—C(═NR_(bb))N(R_(bb))(R_(cc)) or —N(R_(bb))C(═NR_(bb))(R_(aa))), thiol(—SR_(bb)), sulfinyl (—S(O)R_(bb)), sulfonyl (—S(O)₂R_(bb)) andsulfonamidyl (—S(O)₂N(R_(bb))(R_(cc)) or —N(R_(bb))S(O)₂R_(bb)). Whereineach R_(aa), R_(bb) and R_(cc) is, independently, H, an optionallylinked chemical functional group or a further substituent group with apreferred list including without limitation, alkyl, alkenyl, alkynyl,aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic,heterocyclic and heteroarylalkyl. Selected substituents within thecompounds described herein are present to a recursive degree.

As used herein, “alkyl,” as used herein, means a saturated straight orbranched hydrocarbon radical containing up to twenty four carbon atoms.Examples of alkyl groups include without limitation, methyl, ethyl,propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like.Alkyl groups typically include from 1 to about 24 carbon atoms, moretypically from 1 to about 12 carbon atoms (C₁-C₁₂ alkyl) with from 1 toabout 6 carbon atoms being more preferred.

As used herein, “alkenyl,” means a straight or branched hydrocarbonchain radical containing up to twenty four carbon atoms and having atleast one carbon-carbon double bond. Examples of alkenyl groups includewithout limitation, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl,dienes such as 1,3-butadiene and the like. Alkenyl groups typicallyinclude from 2 to about 24 carbon atoms, more typically from 2 to about12 carbon atoms with from 2 to about 6 carbon atoms being morepreferred. Alkenyl groups as used herein may optionally include one ormore further substituent groups.

As used herein, “alkynyl,” means a straight or branched hydrocarbonradical containing up to twenty four carbon atoms and having at leastone carbon-carbon triple bond. Examples of alkynyl groups include,without limitation, ethynyl, 1-propynyl, 1-butynyl, and the like.Alkynyl groups typically include from 2 to about 24 carbon atoms, moretypically from 2 to about 12 carbon atoms with from 2 to about 6 carbonatoms being more preferred. Alkynyl groups as used herein may optionallyinclude one or more further substituent groups.

As used herein, “acyl,” means a radical formed by removal of a hydroxylgroup from an organic acid and has the general Formula —C(O)—X where Xis typically aliphatic, alicyclic or aromatic. Examples includealiphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromaticsulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphaticphosphates and the like. Acyl groups as used herein may optionallyinclude further substituent groups.

As used herein, “alicyclic” means a cyclic ring system wherein the ringis aliphatic. The ring system can comprise one or more rings wherein atleast one ring is aliphatic. Preferred alicyclics include rings havingfrom about 5 to about 9 carbon atoms in the ring. Alicyclic as usedherein may optionally include further substituent groups.

As used herein, “aliphatic” means a straight or branched hydrocarbonradical containing up to twenty four carbon atoms wherein the saturationbetween any two carbon atoms is a single, double or triple bond. Analiphatic group preferably contains from 1 to about 24 carbon atoms,more typically from 1 to about 12 carbon atoms with from 1 to about 6carbon atoms being more preferred. The straight or branched chain of analiphatic group may be interrupted with one or more heteroatoms thatinclude nitrogen, oxygen, sulfur and phosphorus.

Such aliphatic groups interrupted by heteroatoms include withoutlimitation, polyalkoxys, such as polyalkylene glycols, polyamines, andpolyimines. Aliphatic groups as used herein may optionally includefurther substituent groups.

As used herein, “alkoxy” means a radical formed between an alkyl groupand an oxygen atom wherein the oxygen atom is used to attach the alkoxygroup to a parent molecule. Examples of alkoxy groups include withoutlimitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy,tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groupsas used herein may optionally include further substituent groups.

As used herein, “aminoalkyl” means an amino substituted C₁-C₁₂ alkylradical. The alkyl portion of the radical forms a covalent bond with aparent molecule. The amino group can be located at any position and theaminoalkyl group can be substituted with a further substituent group atthe alkyl and/or amino portions.

As used herein, “aralkyl” and “arylalkyl” mean an aromatic group that iscovalently linked to a C₁-C₁₂ alkyl radical. The alkyl radical portionof the resulting aralkyl (or arylalkyl) group forms a covalent bond witha parent molecule. Examples include without limitation, benzyl,phenethyl and the like. Aralkyl groups as used herein may optionallyinclude further substituent groups attached to the alkyl, the aryl orboth groups that form the radical group.

As used herein, “aryl” and “aromatic” mean a mono- or polycycliccarbocyclic ring system radicals having one or more aromatic rings.Examples of aryl groups include without limitation, phenyl, naphthyl,tetrahydronaphthyl, indanyl, idenyl and the like. Preferred aryl ringsystems have from about 5 to about 20 carbon atoms in one or more rings.Aryl groups as used herein may optionally include further substituentgroups.

As used herein, “halo” and “halogen,” mean an atom selected fromfluorine, chlorine, bromine and iodine.

As used herein, “heteroaryl,” and “heteroaromatic,” mean a radicalcomprising a mono- or poly-cyclic aromatic ring, ring system or fusedring system wherein at least one of the rings is aromatic and includesone or more heteroatoms. Heteroaryl is also meant to include fused ringsystems including systems where one or more of the fused rings containno heteroatoms. Heteroaryl groups typically include one ring atomselected from sulfur, nitrogen or oxygen. Examples of heteroaryl groupsinclude without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl,pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl,oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl,benzimidazolyl, benzooxazolyl, quinoxalinyl and the like. Heteroarylradicals can be attached to a parent molecule directly or through alinking moiety such as an aliphatic group or hetero atom. Heteroarylgroups as used herein may optionally include further substituent groups.

As used herein, “conjugate compound” means any atoms, group of atoms, orgroup of linked atoms suitable for use as a conjugate group. In certainembodiments, conjugate compounds may possess or impart one or moreproperties, including, but not limited to pharmacodynamic,pharmacokinetic, binding, absorption, cellular distribution, cellularuptake, charge and/or clearance properties.

As used herein, unless otherwise indicated or modified, the term“double-stranded” refers to two separate oligomeric compounds that arehybridized to one another. Such double stranded compounds may have oneor more or non-hybridizing nucleosides at one or both ends of one orboth strands (overhangs) and/or one or more internal non-hybridizingnucleosides (mismatches) provided there is sufficient complementarity tomaintain hybridization under physiologically relevant conditions.

As used herein, “2′-O-methoxyethyl” (also 2′-MOE and 2′-O(CH₂)₂—OCH₃)refers to an O-methoxyethyl modification of the 2′ position ofa furosylring. A 2′-O-methoxyethyl modified sugar is a modified sugar.

As used herein, “2′-O-methoxyethyl nucleotide” means a nucleotidecomprising a 2′-O-methoxyethyl modified sugar moiety.

“3′ target site” or “3′ stop site” refers to the nucleotide of a targetnucleic acid which is complementary to the 3′-most nucleotide of aparticular antisense compound.

As used herein, “5′ target site” or “5 start site” refers to thenucleotide of a target nucleic acid which is complementary to the5′-most nucleotide of a particular antisense compound.

As used herein, “5-methylcytosine” means a cytosine modified with amethyl group attached to the 5′ position. A 5-methylcytosine is amodified nucleobase.

As used herein, “about” means within +10% of a value. For example, if itis stated, “a marker may be increased by about 50%”, it is implied thatthe marker may be increased between 45%-55% As used herein, “activepharmaceutical agent” means the substance or substances in apharmaceutical composition that provide a therapeutic benefit whenadministered to an individual. For example, in certain embodiments anantisense oligonucleotide targeted to ANGPTL3 is an activepharmaceutical agent.

As used herein, “active target region” or “target region” means a regionto which one or more active antisense compounds is targeted.

As used herein, “active antisense compounds” means antisense compoundsthat reduce target nucleic acid levels or protein levels.

As used herein, “adipogenesis” means the development of fat cells frompreadipocytes. “Lipogenesis” means the production or formation of fat,either fatty degeneration or fatty infiltration.

As used herein, “adiposity” or “obesity” refers to the state of beingobese or an excessively high amount of body fat or adipose tissue inrelation to lean body mass. The amount of body fat includes concern forboth the distribution of fat throughout the body and the size and massof the adipose tissue deposits. Body fat distribution can be estimatedby skin-fold measures, waist-to-hip circumference ratios, or techniquessuch as ultrasound, computed tomography, or magnetic resonance imaging.According to the Center for Disease Control and Prevention, individualswith a body mass index (BMI) of 30 or more are considered obese. Theterm “Obesity” as used herein includes conditions where there is anincrease in body fat beyond the physical requirement as a result ofexcess accumulation of adipose tissue in the body. The term “obesity”includes, but is not limited to, the following conditions: adult-onsetobesity; alimentary obesity; endogenous or metabolic obesity; endocrineobesity; familial obesity; hyperinsulinar obesity;hyperplastic-hypertrophic obesity; hypogonadal obesity; hypothyroidobesity; lifelong obesity; morbid obesity and exogenous obesity.

As used herein, “administered concomitantly” refers to theco-administration of two agents in any manner in which thepharmacological effects of both are manifest in the patient at the sametime. Concomitant administration does not require that both agents beadministered in a single pharmaceutical composition, in the same dosageform, or by the same route of administration. The effects of both agentsneed not manifest themselves at the same time. The effects need only beoverlapping for a period of time and need not be coextensive.

As used herein, “administering” means providing an agent to an animal,and includes, but is not limited to, administering by a medicalprofessional and self-administering.

As used herein, “agent” means an active substance that can provide atherapeutic benefit when administered to an animal. “First Agent” meansa therapeutic compound of the invention. For example, a first agent canbe an antisense oligonucleotide targeting ANGPTL3. “Second agent” meansa second therapeutic compound of the invention (e.g. a second antisenseoligonucleotide targeting ANGPTL3) and/or a non-ANGPTL3 therapeuticcompound.

As used herein, “amelioration” refers to a lessening of at least oneindicator, sign, or symptom of an associated disease, disorder, orcondition. The severity of indicators can be determined by subjective orobjective measures, which are known to those skilled in the art.

As used herein, “ANGPTL3” means any nucleic acid or protein of ANGPTL3.

As used herein, “ANGPTL3 expression” means the level of mRNA transcribedfrom the gene encoding ANGPTL3 or the level of protein translated fromthe mRNA. ANGPTL3 expression can be determined by art known methods suchas a Northern or Western blot.

As used herein, “ANGPTL3 nucleic acid” means any nucleic acid encodingANGPTL3. For example, in certain embodiments, an ANGPTL3 nucleic acidincludes a DNA sequence encoding ANGPTL3, a RNA sequence transcribedfrom DNA encoding ANGPTL3 (including genomic DNA comprising introns andexons), and a mRNA sequence encoding ANGPTL3. “ANGPTL3 mRNA” means amRNA encoding an ANGPTL3 protein.

As used herein, “animal” refers to a human or non-human animal,including, but not limited to, mice, rats, rabbits, dogs, cats, pigs,and non-human primates, including, but not limited to, monkeys andchimpanzees.

As used herein, “apoB-containing lipoprotein” means any lipoprotein thathas apolipoprotein B as its protein component, and is understood toinclude LDL, VLDL, IDL, and lipoprotein(a) and can be generally targetedby lipid lowering agent and therapies. “ApoB-100-containing LDL” meansApoB-100 isoform containing LDL.

As used herein, “atherosclerosis” means a hardening of the arteriesaffecting large and medium-sized arteries and is characterized by thepresence of fatty deposits. The fatty deposits are called “atheromas” or“plaques,” which consist mainly of cholesterol and other fats, calciumand scar tissue, and damage the lining of arteries.

As used herein, “cardiometabolic disease” or “cardiometabolic disorder”are diseases or disorders concerning both the cardiovascular system andthe metabolic system. Examples of cardiometabolic diseases or disordersinclude, but are not limited to, diabetes and dyslipidemias.

As used herein, “co-administration” means administration of two or moreagents to an individual.

The two or more agents can be in a single pharmaceutical composition, orcan be in separate pharmaceutical compositions. Each of the two or moreagents can be administered through the same or different routes ofadministration. Co-administration encompasses parallel or sequentialadministration.

As used herein, “cholesterol” is a sterol molecule found in the cellmembranes of all animal tissues. Cholesterol must be transported in ananimal's blood plasma by lipoproteins including very low densitylipoprotein (VLDL), intermediate density lipoprotein (IDL), low densitylipoprotein (LDL), and high density lipoprotein (HDL). “Plasmacholesterol” refers to the sum of all lipoproteins (VDL, IDL, LDL, HDL)esterified and/or non-estrified cholesterol present in the plasma orserum.

As used herein, “cholesterol absorption inhibitor” means an agent thatinhibits the absorption of exogenous cholesterol obtained from diet.

As used herein, “coronary heart disease (CHD)” means a narrowing of thesmall blood vessels that supply blood and oxygen to the heart, which isoften a result of atherosclerosis.

As used herein, “diabetes mellitus” or “diabetes” is a syndromecharacterized by disordered metabolism and abnormally high blood sugar(hyperglycemia) resulting from insufficient levels of insulin or reducedinsulin sensitivity. The characteristic symptoms are excessive urineproduction (polyuria) due to high blood glucose levels, excessive thirstand increased fluid intake (polydipsia) attempting to compensate forincreased urination, blurred vision due to high blood glucose effects onthe eye's optics, unexplained weight loss, and lethargy.

As used herein, “diabetic dyslipidemia” or “type 2 diabetes withdyslipidemia” means a condition characterized by Type 2 diabetes,reduced HDL-C, elevated triglycerides, and elevated small, dense LDLparticles.

As used herein, “diluent” means an ingredient in a composition thatlacks pharmacological activity, but is pharmaceutically necessary ordesirable. For example, the diluent in an injected composition can be aliquid, e.g. saline solution.

As used herein, “dyslipidemia” refers to a disorder of lipid and/orlipoprotein metabolism, including lipid and/or lipoproteinoverproduction or deficiency. Dyslipidemias may be manifested byelevation of lipids such as cholesterol and triglycerides as well aslipoproteins such as low-density lipoprotein (LDL) cholesterol.

As used herein, “dosage unit” means a form in which a pharmaceuticalagent is provided, e.g. pill, tablet, or other dosage unit known in theart. In certain embodiments, a dosage unit is a vial containinglyophilized antisense oligonucleotide. In certain embodiments, a dosageunit is a vial containing reconstituted antisense oligonucleotide.

As used herein, “dose” means a specified quantity of a pharmaceuticalagent provided in a single administration, or in a specified timeperiod. In certain embodiments, a dose can be administered in one, two,or more boluses, tablets, or injections. For example, in certainembodiments where subcutaneous administration is desired, the desireddose requires a volume not easily accommodated by a single injection,therefore, two or more injections can be used to achieve the desireddose. In certain embodiments, the pharmaceutical agent is administeredby infusion over an extended period of time or continuously. Doses canbe stated as the amount of pharmaceutical agent per hour, day, week, ormonth. Doses can be expressed as mg/kg or g/kg.

As used herein, “effective amount” or “therapeutically effective amount”means the amount of active pharmaceutical agent sufficient to effectuatea desired physiological outcome in an individual in need of the agent.The effective amount can vary among individuals depending on the healthand physical condition of the individual to be treated, the taxonomicgroup of the individuals to be treated, the formulation of thecomposition, assessment of the individual's medical condition, and otherrelevant factors.

As used herein, “glucose” is a monosaccharide used by cells as a sourceof energy and metabolic intermediate. “Plasma glucose” refers to glucosepresent in the plasma.

As used herein, “high density lipoprotein-C(HDL-C)” means cholesterolassociated with high density lipoprotein particles. Concentration ofHDL-C in serum (or plasma) is typically quantified in mg/dL or nmol/L.“serum HDL-C” and “plasma HDL-C” mean HDL-C in serum and plasma,respectively.

As used herein, “HMG-CoA reductase inhibitor” means an agent that actsthrough the inhibition of the enzyme HMG-CoA reductase, such asatorvastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin, andsimvastatin.

As used herein, “hypercholesterolemia” means a condition characterizedby elevated cholesterol or circulating (plasma) cholesterol,LDL-cholesterol and VLDL-cholesterol, as per the guidelines of theExpert Panel Report of the National Cholesterol Educational Program(NCEP) of Detection, Evaluation of Treatment of high cholesterol inadults (see, Arch. Int. Med. (1988) 148, 36-39).

As used herein, “hyperlipidemia” or “hyperlipemia” is a conditioncharacterized by elevated serum lipids or circulating (plasma) lipids.This condition manifests an abnormally high concentration of fats. Thelipid fractions in the circulating blood are cholesterol, low densitylipoproteins, very low density lipoproteins and triglycerides.

As used herein, “hypertriglyceridemia” means a condition characterizedby elevated triglyceride levels.

As used herein, “identifying” or “selecting a subject having a metabolicor cardiovascular disease” means identifying or selecting a subjecthaving been diagnosed with a metabolic disease, a cardiovasculardisease, or a metabolic syndrome; or, identifying or selecting a subjecthaving any symptom of a metabolic disease, cardiovascular disease, ormetabolic syndrome including, but not limited to, hypercholesterolemia,hyperglycemia, hyperlipidemia, hypertriglyceridemia, hypertension,increased insulin resistance, decreased insulin sensitivity, abovenormal body weight, and/or above normal body fat content or anycombination thereof. Such identification may be accomplished by anymethod, including but not limited to, standard clinical tests orassessments, such as measuring serum or circulating (plasma)cholesterol, measuring serum or circulating (plasma) blood-glucose,measuring serum or circulating (plasma) triglycerides, measuringblood-pressure, measuring body fat content, measuring body weight, andthe like.

As used herein, “identifying” or “selecting a diabetic subject” meansidentifying or selecting a subject having been identified as diabetic oridentifying or selecting a subject having any symptom of diabetes (type1 or type 2) such as, but not limited to, having a fasting glucose of atleast 110 mg/dL, glycosuria, polyuria, polydipsia, increased insulinresistance, and/or decreased insulin sensitivity.

As used herein, “identifying” or “selecting an obese subject” meansidentifying or selecting a subject having been diagnosed as obese oridentifying or selecting a subject with a BMI over 30 and/or a waistcircumference of greater than 102 cm in men or greater than 88 cm inwomen.

As used herein, “identifying” or “selecting a subject havingdyslipidemia” means identifying or selecting a subject diagnosed with adisorder of lipid and/or lipoprotein metabolism, including lipid and/orlipoprotein overproduction or deficiency. Dyslipidemias may bemanifested by elevation of lipids such as cholesterol and triglyceridesas well as lipoproteins such as low-density lipoprotein (LDL)cholesterol.

As used herein, “identifying” or “selecting” a subject having increasedadiposity” means identifying or selecting a subject having an increasedamount of body fat (or adiposity) that includes concern for one or boththe distribution of fat throughout the body and the size and mass of theadipose tissue deposits. Body fat distribution can be estimated byskin-fold measures, waist-to-hip circumference ratios, or techniquessuch as ultrasound, computer tomography, or magnetic resonance imaging.According to the Center for Disease Control and Prevention, individualswith a body mass index (BMI) of 30 or more are considered obese.

As used herein, “improved cardiovascular outcome” means a reduction inthe occurrence of adverse cardiovascular events, or the risk thereof.Examples of adverse cardiovascular events include, without limitation,death, reinfarction, stroke, cardiogenic shock, pulmonary edema, cardiacarrest, and atrial dysrhythmia.

As used herein, “immediately adjacent” means there are no interveningelements between the immediately adjacent elements.

As used herein, “individual” or “subject” or “animal” means a human ornon-human animal selected for treatment or therapy.

As used herein, “insulin resistance” is defined as the condition inwhich normal amounts of insulin are inadequate to produce a normalinsulin response from cells, e.g., fat, muscle and/or liver cells.Insulin resistance in fat cells results in hydrolysis of storedtriglycerides, which elevates free fatty acids in the blood plasma.Insulin resistance in muscle reduces glucose uptake whereas insulinresistance in liver reduces glucose storage, with both effects servingto elevate blood glucose. High plasma levels of insulin and glucose dueto insulin resistance often leads to metabolic syndrome and type 2diabetes.

As used herein, “insulin sensitivity” is a measure of how effectively anindividual processes glucose.

An individual having high insulin sensitivity effectively processesglucose whereas an individual with low insulin sensitivity does noteffectively process glucose.

As used herein, “intravenous administration” means administration into avein.

As used herein, “lipid-lowering” means a reduction in one or more lipidsin a subject. Lipid-lowering can occur with one or more doses over time.

As used herein, “lipid-lowering agent” means an agent, for example, anANGPTL3-specific modulator, provided to a subject to achieve a loweringof lipids in the subject. For example, in certain embodiments, alipid-lowering agent is provided to a subject to reduce one or more ofapoB, apoC-III, total cholesterol, LDL-C, VLDL-C, IDL-C, non-HDL-C,triglycerides, small dense LDL particles, and Lp(a) in a subject.

As used herein, “lipid-lowering therapy” means a therapeutic regimenprovided to a subject to reduce one or more lipids in a subject. Incertain embodiments, a lipid-lowering therapy is provided to reduce oneor more of apoB, apoC-III, total cholesterol, LDL-C, VLDL-C, IDL-C,non-HDL-C, triglycerides, small dense LDL particles, and Lp(a) in asubject.

As used herein, “lipoprotein”, such as VLDL, LDL and HDL, refers to agroup of proteins found in the serum, plasma and lymph and are importantfor lipid transport. The chemical composition of each lipoproteindiffers in that the HDL has a higher proportion of protein versus lipid,whereas the VLDL has a lower proportion of protein versus lipid.

As used herein, “low density lipoprotein-cholesterol (LDL-C)” meanscholesterol carried in low density lipoprotein particles. Concentrationof LDL-C in serum (or plasma) is typically quantified in mg/dL ornmol/L. “Serum LDL-C” and “plasma LDL-C” mean LDL-C in the serum andplasma, respectively.

As used herein, “major risk factors” refers to factors that contributeto a high risk for a particular disease or condition. In certainembodiments, major risk factors for coronary heart disease include,without limitation, cigarette smoking, hypertension, low HDL-C, familyhistory of coronary heart disease, age, and other factors disclosedherein.

As used herein, “metabolic disorder” or “metabolic disease” refers to acondition characterized by an alteration or disturbance in metabolicfunction. “Metabolic” and “metabolism” are terms well known in the artand generally include the whole range of biochemical processes thatoccur within a living organism. Metabolic disorders include, but are notlimited to, hyperglycemia, prediabetes, diabetes (type I and type 2),obesity, insulin resistance, metabolic syndrome and dyslipidemia due totype 2 diabetes.

As used herein, “metabolic syndrome” means a condition characterized bya clustering of lipid and non-lipid cardiovascular risk factors ofmetabolic origin. In certain embodiments, metabolic syndrome isidentified by the presence of any 3 of the following factors: waistcircumference of greater than 102 cm in men or greater than 88 cm inwomen; serum triglyceride of at least 150 mg/dL; HDL-C less than 40mg/dL in men or less than 50 mg/dL in women; blood pressure of at least130/85 mmHg; and fasting glucose of at least 110 mg/dL. Thesedeterminants can be readily measured in clinical practice (JAMA, 2001,285: 2486-2497).

As used herein, “mixed dyslipidemia” means a condition characterized byelevated cholesterol and elevated triglycerides.

As used herein, “MTP inhibitor” means an agent inhibits the enzymemicrosomal triglyceride transfer protein.

As used herein, “non-alcoholic fatty liver disease” or “NAFLD” means acondition characterized by fatty inflammation of the liver that is notdue to excessive alcohol use (for example, alcohol consumption of over20 g/day). In certain embodiments, NAFLD is related to insulinresistance and metabolic syndrome. NAFLD encompasses a disease spectrumranging from simple triglyceride accumulation in hepatocytes (hepaticsteatosis) to hepatic steatosis with inflammation (steatohepatitis),fibrosis, and cirrhosis.

As used herein, “nonalcoholic steatohepatitis” (NASH) occurs fromprogression of NAFLD beyond deposition of triglycerides. A “second hit”capable of inducing necrosis, inflammation, and fibrosis is required fordevelopment of NASH. Candidates for the second-hit can be grouped intobroad categories: factors causing an increase in oxidative stress andfactors promoting expression of proinflammatory cytokines. It has beensuggested that increased liver triglycerides lead to increased oxidativestress in hepatocytes of animals and humans, indicating a potentialcause-and-effect relationship between hepatic triglyceride accumulation,oxidative stress, and the progression of hepatic steatosis to NASH(Browning and Horton, J Clin Invest, 2004, 114, 147-152).Hypertriglyceridemia and hyperfattyacidemia can cause triglycerideaccumulation in peripheral tissues (Shimamura et al., Biochem BiophysRes Commun, 2004, 322, 1080-1085).

As used herein, “nucleic acid” refers to molecules composed of monomericnucleotides. A nucleic acid includes ribonucleic acids (RNA),deoxyribonucleic acids (DNA), single-stranded nucleic acids,double-stranded nucleic acids, small interfering ribonucleic acids(siRNA), and microRNAs (miRNA). A nucleic acid can also comprise acombination of these elements in a single molecule.

As used herein, “parenteral administration” means administration by amanner other than through the digestive tract. Parenteral administrationincludes topical administration, subcutaneous administration,intravenous administration, intramuscular administration, intraarterialadministration, intraperitoneal administration, or intracranialadministration, e.g. intrathecal or intracerebroventricularadministration. Administration can be continuous, or chronic, or shortor intermittent.

As used herein, “pharmaceutical agent” means a substance that provides atherapeutic benefit when administered to an individual. For example, incertain embodiments, an antisense oligonucleotide targeted to ANGPTL3 ispharmaceutical agent.

As used herein, “pharmaceutical composition” means a mixture ofsubstances suitable for administering to an individual. For example, apharmaceutical composition can comprise one or more active agents and asterile aqueous solution.

As used herein, “pharmaceutically acceptable carrier” means a medium ordiluent that does not interfere with the structure or function of theoligonucleotide. Certain, of such carries enable pharmaceuticalcompositions to be formulated as, for example, tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspension and lozenges forthe oral ingestion by a subject. Certain of such carriers enablepharmaceutical compositions to be formulated for injection or infusion.For example, a pharmaceutically acceptable carrier can be a sterileaqueous solution.

As used herein, “pharmaceutically acceptable salts” meansphysiologically and pharmaceutically acceptable salts of antisensecompounds, i.e., salts that retain the desired biological activity ofthe parent oligonucleotide and do not impart undesired toxicologicaleffects thereto.

As used herein, “portion” means a defined number of contiguous (i.e.linked) nucleobases of a nucleic acid. In certain embodiments, a portionis a defined number of contiguous nucleobases of a target nucleic acid.In certain embodiments, a portion is a defined number of contiguousnucleobases of an antisense compound.

As used herein, “prevent” refers to delaying or forestalling the onsetor development of a disease, disorder, or condition for a period of timefrom minutes to indefinitely. Prevent also means reducing risk ofdeveloping a disease, disorder, or condition.

As used herein, “side effects” means physiological responsesattributable to a treatment other than the desired effects. In certainembodiments, side effects include injection site reactions, liverfunction test abnormalities, renal function abnormalities, livertoxicity, renal toxicity, central nervous system abnormalities,myopathies, and malaise. For example, increased aminotransferase levelsin serum can indicate liver toxicity or liver function abnormality. Forexample, increased bilirubin can indicate liver toxicity or liverfunction abnormality.

As used herein, “statin” means an agent that inhibits the activity ofHMG-CoA reductase.

As used herein, “subcutaneous administration” means administration justbelow the skin.

As used herein, “targeting” or “targeted” means the process of designand selection of an antisense compound that will specifically hybridizeto a target nucleic acid and induce a desired effect.

As used herein, “target nucleic acid,” “target RNA,” and “target RNAtranscript” all refer to a nucleic acid capable of being targeted byantisense compounds.

As used herein, “target region” is defined as a portion of the targetnucleic acid having at least one identifiable structure, function, orcharacteristic.

As used herein, “target segment” means the sequence of nucleotides of atarget nucleic acid to which one or more antisense compound is targeted.“5′ target site” or “5′ start site” refers to the 5′-most nucleotide ofa target segment. “3′ target site” or “3′ stop site” refers to the3′-most nucleotide of a target segment.

As used herein, “therapeutically effective amount” means an amount of anagent that provides a therapeutic benefit to an individual.

As used herein, “therapeutic lifestyle change” means dietary andlifestyle changes intended to lower fat/adipose tissue mass and/orcholesterol. Such change can reduce the risk of developing heartdisease, and may include recommendations for dietary intake of totaldaily calories, total fat, saturated fat, polyunsaturated fat,monounsaturated fat, carbohydrate, protein, cholesterol, insolublefiber, as well as recommendations for physical activity.

As used herein, “triglyceride” means a lipid or neutral fat consistingof glycerol combined with three fatty acid molecules.

As used herein, “type 2 diabetes” (also known as “type 2 diabetesmellitus” or “diabetes mellitus, type 2”, and formerly called “diabetesmellitus type 2”, “non-insulin-dependent diabetes (NIDDM)”, “obesityrelated diabetes”, or “adult-onset diabetes”) is a metabolic disorderthat is primarily characterized by insulin resistance, relative insulindeficiency, and hyperglycemia.

As used herein, “treat” refers to administering a pharmaceuticalcomposition to effect an alteration or improvement of a disease,disorder, or condition.

Certain Embodiments

In certain embodiments disclosed herein, ANGPTL3 has the sequence as setforth in GenBank Accession No. NM_014495.2 (incorporated herein as SEQID NO: 1). In certain embodiments, ANGPTL3 has the sequence as set forthin GenBank Accession No. NT_032977.9 nucleotides 33032001 to 33046000(incorporated herein as SEQ ID NO: 2).

Certain embodiments disclosed herein provide compounds or compositionscomprising a modified oligonucleotide and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 nucleosides having anucleobase sequence comprising at least 8 contiguous nucleobasescomplementary to an equal length portion of SEQ ID NOs: 1-2.

In certain embodiments, a compound comprises a siRNA or antisenseoligonucleotide targeted to ANGPTL3 known in the art and a conjugategroup described herein. Examples of antisense oligonucleotides targetedto ANGPTL3 suitable for conjugation include but are not limited to thosedisclosed in U.S. Pat. No. 8,653,047 (WO 2011/085271), which isincorporated by reference in its entirety herein. In certainembodiments, a compound comprises an antisense oligonucleotide having anucleobase sequence of any of SEQ ID NOs: 34-111 disclosed in U.S. Pat.No. 8,653,047 and a conjugate group described herein. In certainembodiments, a compound comprises a siRNA sense or antisense strandhaving a nucleobase sequence of any of SEQ ID NOs: 34-111 disclosed inU.S. Pat. No. 8,653,047 and a conjugate group described herein. Thenucleobase sequences of all of the aforementioned referenced SEQ ID NOsare incorporated by reference herein.

Certain embodiments disclosed herein provide compounds or compositionscomprising a modified oligonucleotide and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides inlength targeted to ANGPTL3. The ANGPTL3 target can have a sequenceselected from any one of SEQ ID NOs: 1-2.

Certain embodiments disclosed herein provide compounds or compositionscomprising a modified oligonucleotide and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides andcomprising a nucleobase sequence comprising a portion of at least 8contiguous nucleobases complementary to an equal length portion ofnucleobases 1140 to 1159 of SEQ ID NO: 1, wherein the nucleobasesequence of the modified oligonucleotide is at least 80% complementaryto SEQ ID NO: 1. In certain embodiments, the modified oligonucleotide isat least 8, least 9, least 10, least 11, at least 12, least 13, at least14, at least 15, at least 16, least 17, least 18, least 19, or 20contiguous nucleobases complementary to an equal length portion ofnucleobases 1140 to 1159 of SEQ ID NO: 1.

Certain embodiments disclosed herein provide compounds or compositionscomprising a modified oligonucleotide and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides andcomprising a nucleobase sequence complementary to nucleobases 1140 to1159 of SEQ ID NO: 1, wherein the nucleobase sequence of the modifiedoligonucleotide is at least 80% complementary to SEQ ID NO: 1.

Certain embodiments disclosed herein provide compounds or compositionscomprising a modified oligonucleotide and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides andcomprising a nucleobase sequence comprising a portion of at least 8contiguous nucleobases complementary to an equal length portion ofnucleobases 1907 to 1926 of SEQ ID NO: 1, wherein the nucleobasesequence of the modified oligonucleotide is at least 80% complementaryto SEQ ID NO: 1. In certain embodiments, the modified oligonucleotide isat least 8, least 9, least 10, least 11, at least 12, least 13, at least14, at least 15, at least 16, least 17, least 18, least 19, or 20contiguous nucleobases complementary to an equal length portion ofnucleobases 1907 to 1926 of SEQ ID NO: 1.

Certain embodiments disclosed herein provide compounds or compositionscomprising a modified oligonucleotide and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides andcomprising a nucleobase sequence complementary to nucleobases 1907 to1926 of SEQ ID NO: 1, wherein the nucleobase sequence of the modifiedoligonucleotide is at least 80% complementary to SEQ ID NO: 1.

Certain embodiments disclosed herein provide compounds or compositionscomprising a modified oligonucleotide and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides andcomprising a nucleobase sequence comprising a portion of at least 8contiguous nucleobases complementary to an equal length portion ofnucleobases 147 to 162 of SEQ ID NO: 1, wherein the nucleobase sequenceof the modified oligonucleotide is at least 80% complementary to SEQ IDNO: 1. In certain embodiments, the modified oligonucleotide is at least8, least 9, least 10, least 11, at least 12, least 13, at least 14, atleast 15, or 16 contiguous nucleobases complementary to an equal lengthportion of nucleobases 147 to 162 of SEQ ID NO: 1.

Certain embodiments disclosed herein provide compounds or compositionscomprising a modified oligonucleotide and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides andcomprising a nucleobase sequence complementary to nucleobases 147 to 162of SEQ ID NO: 1, wherein the nucleobase sequence of the modifiedoligonucleotide is at least 80% complementary to SEQ ID NO: 1.

In certain embodiments, the modified oligonucleotide consists of 12 to30, 15 to 30, 18 to 24, 19 to 22, 13 to 25, 14 to 25, 15 to 25 or 16 to24 linked nucleosides. In certain embodiments, the modifiedoligonucleotide consists of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 linked nucleosides or arange defined by any two of these values. In certain embodiments, themodified oligonucleotide is 16 linked nucleosides in length. In certainembodiments, the modified oligonucleotide is 20 linked nucleosides inlength.

In certain embodiments, the modified oligonucleotide comprises anucleobase sequence comprising a portion of at least 8, least 9, least10, least 11, at least 12, least 13, at least 14, at least 15, at least16, least 17, least 18, least 19, or 20 contiguous nucleobasescomplementary to an equal length portion of SEQ ID NO: 1 or 2.

Certain embodiments disclosed herein provide compounds or compositionscomprising a modified oligonucleotide and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides andhaving a nucleobase sequence comprising at least 8, least 9, least 10,least 11, at least 12, least 13, at least 14, at least 15, at least 16,least 17, least 18, least 19, or 20 contiguous nucleobases of anucleobase sequence selected from any one of SEQ ID NOs: 15-27, 30-73,75-85, 87-232, 238, 240-243, 245-247, 249-262, 264-397, 399-469,471-541, 543-600, 604-760, 762-819, 821-966, 968-971, 973-975, 977-990,992-1110, 1112-1186, 1188-1216, 1218-1226, 1228-1279, 1281-1293,1295-1304, 1306-1943, 1945-1951, 1953-1977, 1979-1981, 1983-2044,2046-2097, 2099-2181, 2183-2232, 2234-2238, 2240-2258, 2260-2265,2267-2971, 2973-2976, 2978-4162, 4164-4329, 4331-4389, 4391-4394,4396-4877.

Certain embodiments disclosed herein provide compounds or compositionscomprising a modified oligonucleotide and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides andhaving a nucleobase sequence comprising at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, or 20 contiguousnucleobases of the nucleobase sequences of SEQ ID NO: 77. In certainembodiments, the compound comprises ISIS 563580 and a conjugate group.In certain embodiments, the compound consists of ISIS 563580 and aconjugate group.

In certain embodiments, the present disclosure provides conjugatedantisense compounds represented by the following structure. In certainembodiments, the antisense compound comprises the modifiedoligonucleotide ISIS 563580 with a 5′-X, wherein X is a conjugate groupcomprising GalNAc. In certain embodiments, the antisense compoundconsists of the modified oligonucleotide ISIS 563580 with a 5′-X,wherein X is a conjugate group comprising GalNAc.

In certain embodiments, the present disclosure provides conjugatedantisense compounds represented by the following structure. In certainembodiments, the antisense compound comprises the conjugated modifiedoligonucleotide ISIS 703801. In certain embodiments, the antisensecompound consists of the conjugated modified oligonucleotide ISIS703801.

In certain embodiments, the present disclosure provides conjugatedantisense compounds represented by the following structure. In certainembodiments, the antisense compound comprises the conjugated modifiedoligonucleotide ISIS 703802. In certain embodiments, the antisensecompound consists of the conjugated modified oligonucleotide ISIS703802.

In certain embodiments, the present disclosure provides conjugatedantisense compounds represented by the following structure. In certainembodiments, the antisense compound comprises a modified oligonucleotidewith the nucleobase sequence of SEQ ID NO: 77 with a 5′-GalNAc withvariability in the sugar mods of the wings. In certain embodiments, theantisense compound consists of a modified oligonucleotide with thenucleobase sequence of SEQ ID NO: 77 with a 5′-GalNAc with variabilityin the sugar mods of the wings.

wherein either R¹ is —OCH₂CH₂OCH₃ (MOE) and R² is H; or R¹ and R²together form a bridge, wherein R¹ is —O— and R² is —CH₂—, —CH(CH₃)—, or—CH₂CH₂—, and R¹ and R² are directly connected such that the resultingbridge is selected from: —O—CH₂—, —O—CH(CH₃)—, and —O—CH₂CH₂—;

and for each pair of R³ and R⁴ on the same ring, independently for eachring: either R³ is selected from H and —OCH₂CH₂OCH₃ and R⁴ is H; or R³and R⁴ together form a bridge, wherein R³ is —O—, and R⁴ is —CH₂—,—CH(CH₃)—, or —CH₂CH₂— and R³ and R⁴ are directly connected such thatthe resulting bridge is selected from: —O—CH₂—, —O—CH(CH₃)—, and—O—CH₂CH₂—;

and R⁵ is selected from H and —CH₃;

and Z is selected from S— and O—.

Certain embodiments disclosed herein provide compounds or compositionscomprising a modified oligonucleotide and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides andhaving a nucleobase sequence comprising at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, or 20 contiguousnucleobases of the nucleobase sequence of SEQ ID NO: 20. In certainembodiments, the compound comprises ISIS 544199 and a conjugate group.In certain embodiments, the compound consists of ISIS 544199 and aconjugate group.

Certain embodiments disclosed herein provide compounds or compositionscomprising a modified oligonucleotide and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides andhaving a nucleobase sequence comprising at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, or 20 contiguousnucleobases of the nucleobase sequence of SEQ ID NO: 35. In certainembodiments, the compound comprises ISIS 560400 and a conjugate group.In certain embodiments, the compound consists of ISIS 560400 and aconjugate group.

Certain embodiments disclosed herein provide compounds or compositionscomprising a modified oligonucleotide and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides andhaving a nucleobase sequence comprising at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, or 20 contiguousnucleobases of the nucleobase sequence of SEQ ID NO: 90. In certainembodiments, the compound comprises ISIS 567233 and a conjugate group.In certain embodiments, the compound consists of ISIS 567233 and aconjugate group.

Certain embodiments disclosed herein provide compounds or compositionscomprising a modified oligonucleotide and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides andhaving a nucleobase sequence comprising at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, or 20 contiguousnucleobases of the nucleobase sequence of SEQ ID NO: 93. In certainembodiments, the compound comprises ISIS 567320 and a conjugate group.In certain embodiments, the compound consists of ISIS 567320 and aconjugate group.

Certain embodiments disclosed herein provide compounds or compositionscomprising a modified oligonucleotide and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides andhaving a nucleobase sequence comprising at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, or 20 contiguousnucleobases of the nucleobase sequence of SEQ ID NO: 94. In certainembodiments, the compound comprises ISIS 567321 and a conjugate group.In certain embodiments, the compound consists of ISIS 567321 and aconjugate group.

Certain embodiments disclosed herein provide compounds or compositionscomprising a modified oligonucleotide and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides andhaving a nucleobase sequence comprising at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15, or16 contiguous nucleobases of the nucleobase sequence of SEQ ID NO: 110.In certain embodiments, the compound comprises ISIS 559277 and aconjugate group. In certain embodiments, the compound consists of ISIS559277 and a conjugate group.

Certain embodiments disclosed herein provide compounds or compositionscomprising a modified oligonucleotide and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides andhaving a nucleobase sequence comprising at least 8, at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15, or16 contiguous nucleobases of the nucleobase sequence of SEQ ID NO: 114.In certain embodiments, the compound comprises ISIS 561011 and aconjugate group. In certain embodiments, the compound consists of ISIS561011 and a conjugate group.

In certain embodiments, the nucleobase sequence of the modifiedoligonucleotide is at least 70%, 75%, 80%, 85%, 90%, 95% or 100%complementary to any one of SEQ ID NO: 1-2 as measured over the entiretyof the modified oligonucleotide.

In certain embodiments, the compound disclosed herein is asingle-stranded oligonucleotide. In certain embodiments, the compounddisclosed herein is a single-stranded modified oligonucleotide.

In certain embodiments, at least one internucleoside linkage of saidmodified oligonucleotide is a modified internucleoside linkage. Incertain embodiments, the modified internucleoside linkage is aphosphorothioate internucleoside linkage. In certain embodiments, atleast 1, at least 2, at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9 or at least 10 internucleoside linkagesof said modified oligonucleotide are phosphorothioate internucleosidelinkages. In certain embodiments, each internucleoside linkage is aphosphorothioate internucleoside linkage. In certain embodiments, themodified oligonucleotide comprises at least 1, at least 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9or at least 10 phosphodiester internucleoside linkages. In certainembodiments, each internucleoside linkage of the modifiedoligonucleotide is selected from a phosphodiester internucleosidelinkage and a phosphorothioate internucleoside linkage.

In certain embodiments, at least one nucleoside of the modifiedoligonucleotide comprises a modified sugar. In certain embodiments, atleast one modified sugar is a bicyclic sugar. In certain embodiments, atleast one modified sugar comprises a 2′-O-methoxyethyl, a constrainedethyl, a 3′-fluoro-HNA or a 4′-(CH₂)_(n)—O-2′ bridge, wherein n is 1 or2.

In certain embodiments, at least one nucleoside of said modifiedoligonucleotide comprises a modified nucleobase. In certain embodiments,the modified nucleobase is a 5-methylcytosine.

Certain embodiments disclosed herein provide compounds or compositionscomprising a modified oligonucleotide and a conjugate group, wherein themodified oligonucleotide has: a) a gap segment consisting of linkeddeoxynucleosides; b) a 5′ wing segment consisting of linked nucleosides;and c) a 3′ wing segment consisting of linked nucleosides. The gapsegment is positioned between the 5′ wing segment and the 3′ wingsegment and each nucleoside of each wing segment comprises a modifiedsugar.

In certain embodiments, the modified oligonucleotide consists of 12 to30 linked nucleosides and comprises: a gap segment consisting of linkeddeoxynucleosides; a 5′ wing segment consisting of linked nucleosides; a3′ wing segment consisting of linked nucleosides; wherein the gapsegment is positioned between the 5′ wing segment and the 3′ wingsegment and wherein each nucleoside of each wing segment comprises amodified sugar.

In certain embodiments, the compounds or compositions disclosed hereincomprise a modified oligonucleotide consisting of 20 linked nucleosideshaving a nucleobase sequence comprising at least 8 contiguousnucleobases complementary to an equal length portion of SEQ ID NO: 1-2,wherein the modified oligonucleotide comprises: a gap segment consistingof ten linked deoxynucleosides; a 5′ wing segment consisting of fivelinked nucleosides; and a 3′ wing segment consisting of five linkednucleosides; wherein the gap segment is positioned between the 5′ wingsegment and the 3′ wing segment; wherein each nucleoside of each wingsegment comprises a 2′-O-methoxyethyl sugar; wherein at least oneinternucleoside linkage is a phosphorothioate linkage and wherein eachcytosine residue is a 5-methylcytosine. In certain embodiments, eachinternucleoside linkage is a phosphorothioate linkage.

In certain embodiments, the modified oligonucleotide consists of 20linked nucleosides and comprises: a gap segment consisting of ten linkeddeoxynucleosides; a 5′ wing segment consisting of five linkednucleosides; a 3′ wing segment consisting of five linked nucleosides;wherein the gap segment is positioned between the 5′ wing segment andthe 3′ wing segment; wherein each nucleoside of each wing segmentcomprises a 2′-O-methoxyethyl sugar; wherein at least oneinternucleoside linkage is a phosphorothioate linkage and wherein eachcytosine residue is a 5-methylcytosine. In certain embodiments, eachinternucleoside linkage is a phosphorothioate linkage.

In certain embodiments, the compounds or compositions disclosed hereincomprise a modified oligonucleotide consisting of 20 linked nucleosideshaving a nucleobase sequence comprising at least 8 contiguousnucleobases of a nucleobase sequence selected of SEQ ID NO: 77, whereinthe modified oligonucleotide comprises: a gap segment consisting of tenlinked deoxynucleosides; a 5′ wing segment consisting of five linkednucleosides; and a 3′ wing segment consisting of five linkednucleosides; wherein the gap segment is positioned between the 5′ wingsegment and the 3′ wing segment; wherein each nucleoside of each wingsegment comprises a 2′-O-methoxyethyl sugar; wherein at least oneinternucleoside linkage is a phosphorothioate linkage and wherein eachcytosine residue is a 5-methylcytosine. In certain embodiments, eachinternucleoside linkage is a phosphorothioate linkage.

In certain embodiments, the modified oligonucleotide consists of 20linked nucleosides with the nucleobase sequence of SEQ ID NO: 77 andcomprises: a gap segment consisting often linked deoxynucleosides; a 5′wing segment consisting of five linked nucleosides; a 3′ wing segmentconsisting of five linked nucleosides; wherein the gap segment ispositioned between the 5′ wing segment and the 3′ wing segment; whereineach nucleoside of each wing segment comprises a 2′-O-methoxyethylsugar; wherein at least one internucleoside linkage is aphosphorothioate linkage and wherein each cytosine residue is a5-methylcytosine. In certain embodiments, each internucleoside linkageis a phosphorothioate linkage.

In certain embodiments, the compounds or compositions disclosed hereincomprise a modified oligonucleotide consisting of 20 linked nucleosideshaving a nucleobase sequence comprising at least 8 contiguousnucleobases of a nucleobase sequence selected of SEQ ID NO: 20, whereinthe modified oligonucleotide comprises: a gap segment consisting of tenlinked deoxynucleosides; a 5′ wing segment consisting of five linkednucleosides; and a 3′ wing segment consisting of five linkednucleosides; wherein the gap segment is positioned between the 5′ wingsegment and the 3′ wing segment; wherein each nucleoside of each wingsegment comprises a 2′-O-methoxyethyl sugar; wherein at least oneinternucleoside linkage is a phosphorothioate linkage and wherein eachcytosine residue is a 5-methylcytosine. In certain embodiments, eachinternucleoside linkage is a phosphorothioate linkage.

In certain embodiments, the modified oligonucleotide consists of 20linked nucleosides with the nucleobase sequence of SEQ ID NO: 20 andcomprises: a gap segment consisting often linked deoxynucleosides; a 5′wing segment consisting of five linked nucleosides; a 3′ wing segmentconsisting of five linked nucleosides; wherein the gap segment ispositioned between the 5′ wing segment and the 3′ wing segment; whereineach nucleoside of each wing segment comprises a 2′-O-methoxyethylsugar; wherein at least one internucleoside linkage is aphosphorothioate linkage and wherein each cytosine residue is a5-methylcytosine. In certain embodiments, each internucleoside linkageis a phosphorothioate linkage.

In certain embodiments, the compounds or compositions disclosed hereincomprise a modified oligonucleotide consisting of 16 linked nucleosideshaving a nucleobase sequence comprising at least 8 contiguousnucleobases of a nucleobase sequence of SEQ ID NO: 110, wherein themodified oligonucleotide comprises: a gap segment consisting of tenlinked deoxynucleosides; a 5′ wing segment consisting of three linkednucleosides; and a 3′ wing segment consisting of three linkednucleosides; wherein the gap segment is positioned between the 5′ wingsegment and the 3′ wing segment; wherein each wing segment comprises atleast one 2′-O-methoxyethyl sugar and at least one cEt sugar; wherein atleast one internucleoside linkage is a phosphorothioate linkage andwherein each cytosine residue is a 5-methylcytosine. In certainembodiments, each internucleoside linkage is a phosphorothioate linkage.

In certain embodiments, the modified oligonucleotide consists of 16linked nucleosides with the nucleobase sequence of SEQ ID NO: 110 andcomprises: a gap segment consisting of ten linked deoxynucleosides; a 5′wing segment consisting of three linked nucleosides; a 3′ wing segmentconsisting of three linked nucleosides; wherein the gap segment ispositioned between the 5′ wing segment and the 3′ wing segment; whereineach wing segment comprises at least one 2′-O-methoxyethyl sugar and atleast one cEt sugar; wherein at least one internucleoside linkage is aphosphorothioate linkage and wherein each cytosine residue is a5-methylcytosine. In certain embodiments, each internucleoside linkageis a phosphorothioate linkage.

In certain embodiments, the conjugate group is linked to the modifiedoligonucleotide at the 5′ end of the modified oligonucleotide. Incertain embodiments, the conjugate group is linked to the modifiedoligonucleotide at the 3′ end of the modified oligonucleotide.

In certain embodiments, the conjugate group comprises exactly oneligand. In certain embodiments, the conjugate group comprises one ormore ligands. In certain embodiments, the conjugate group comprisesexactly two ligands. In certain embodiments, the conjugate groupcomprises two or more ligands. In certain embodiments, the conjugategroup comprises three or more ligands. In certain embodiments, theconjugate group comprises exactly three ligands. In certain embodiments,each ligand is selected from among: a polysaccharide, modifiedpolysaccharide, mannose, galactose, a mannose derivative, a galactosederivative, D-mannopyranose, L-Mannopyranose, D-Arabinose, L-Galactose,D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-Galactose,L-Galactose, α-D-Mannofuranose, β-D-Mannofuranose, α-D-Mannopyranose,β-D-Mannopyranose, α-D-Glucopyranose, β-D-Glucopyranose,α-D-Glucofuranose, β-D-Glucofuranose, α-D-fructofuranose,α-D-fructopyranose, α-D-Galactopyranose, β-D-Galactopyranose,α-D-Galactofuranose, β-D-Galactofuranose, glucosamine, sialic acid,α-D-galactosamine, N-Acetylgalactosamine,2-Amino-3-O—[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose,2-Deoxy-2-methylamino-L-glucopyranose,4,6-Dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose,2-Deoxy-2-sulfoamino-D-glucopyranose, N-Glycoloyl-α-neuraminic acid,5-thio-β-D-glucopyranose, methyl2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside,4-Thio-β-D-galactopyranose, ethyl3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-gluco-heptopyranoside,2,5-Anhydro-D-allononitrile, ribose, D-ribose, D-4-thioribose, L-ribose,L-4-thioribose. In certain embodiments, each ligand is N-acetylgalactosamine.

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises at least onephosphorus linking group or neutral linking group.

In certain embodiments, the conjugate group comprises a structureselected from among:

-   -   wherein n is from 1 to 12; and    -   wherein m is from 1 to 12.

In certain embodiments, the conjugate group has a tether having astructure selected from among:

wherein L is either a phosphorus linking group or a neutral linkinggroup;

-   -   Z₁ is C(═O)O—R₂;    -   Z₂ is H, C₁-C₆ alkyl or substituted C₁-C₆ alky;    -   R₂ is H, C₁-C₆ alkyl or substituted C₁-C₆ alky; and    -   each m₁ is, independently, from 0 to 20 wherein at least one m₁        is greater than 0 for each tether.

In certain embodiments, the conjugate group has a tether having astructure selected from among:

-   -   wherein Z₂ is H or CH₃; and    -   each m₁ is, independently, from 0 to 20 wherein at least one m₁        is greater than 0 for each tether.

In certain embodiments, the conjugate group has tether having astructure selected from among:

-   -   wherein n is from 1 to 12; and    -   wherein m is from 1 to 12.

In certain embodiments, the conjugate group is covalently attached tothe modified oligonucleotide.

In certain embodiments, the compound has a structure represented by theformula:

A-B—C-DE-F)_(q)

-   -   wherein    -   A is the modified oligonucleotide;    -   B is the cleavable moiety    -   C is the conjugate linker    -   D is the branching group    -   each E is a tether;    -   each F is a ligand; and    -   q is an integer between 1 and 5.

In certain embodiments, the compound has a structure represented by theformula:

AB_(n) ₂ C_(n) ₁ D_(n) ₃ E-F_(q)

-   -   wherein:    -   A is the modified oligonucleotide;    -   B is the cleavable moiety    -   C is the conjugate linker    -   D is the branching group    -   each E is a tether;    -   each F is a ligand;    -   each n is independently 0 or 1; and    -   q is an integer between 1 and 5.

In certain embodiments, the compound has a structure represented by theformula:

A-B—CE-F)_(q)

-   -   wherein    -   A is the modified oligonucleotide;    -   B is the cleavable moiety;    -   C is the conjugate linker;    -   each E is a tether;    -   each F is a ligand; and    -   q is an integer between 1 and 5.

In certain embodiments, the compound has a structure represented by theformula:

A-C-D-E-F)

-   -   wherein    -   A is the modified oligonucleotide;    -   C is the conjugate linker;    -   D is the branching group;    -   each E is a tether;    -   each F is a ligand; and    -   q is an integer between 1 and 5.

In certain embodiments, the compound has a structure represented by theformula:

A-C-E-F)_(q)

-   -   wherein    -   A is the modified oligonucleotide;    -   C is the conjugate linker;    -   each E is a tether;    -   each F is a ligand; and    -   q is an integer between 1 and 5.

In certain embodiments, the compound has a structure represented by theformula:

A-B-DE-F)

-   -   wherein    -   A is the modified oligonucleotide;    -   B is the cleavable moiety;    -   D is the branching group;    -   each E is a tether;    -   each F is a ligand; and    -   q is an integer between 1 and 5.

In certain embodiments, the compound has a structure represented by theformula:

A-B-DE-F)_(q)

-   -   wherein    -   A is the modified oligonucleotide;    -   B is the cleavable moiety;    -   each E is a tether;    -   each F is a ligand; and    -   q is an integer between 1 and 5.

In certain embodiments, the compound has a structure represented by theformula:

A-DE-F)_(q)

-   -   wherein    -   A is the modified oligonucleotide;    -   D is the branching group;    -   each E is a tether;    -   each F is a ligand; and    -   q is an integer between 1 and 5.

In certain embodiments, the conjugate linker has a structure selectedfrom among:

wherein each L is, independently, a phosphorus linking group or aneutral linking group; and

each n is, independently, from 1 to 20.

In certain embodiments, the conjugate linker has a structure selectedfrom among:

In certain embodiments, the conjugate linker has the followingstructure:

In certain embodiments, the conjugate linker has a structure selectedfrom among:

In certain embodiments, the conjugate linker has a structure selectedfrom among:

In certain embodiments, the conjugate linker has a structure selectedfrom among:

In certain embodiments, the conjugate linker comprises a pyrrolidine. Incertain embodiments, the conjugate linker does not comprise apyrrolidine.

In certain embodiments, the conjugate linker comprises PEG.

In certain embodiments, the conjugate linker comprises an amide. Incertain embodiments, the conjugate linker comprises at least two amides.In certain embodiments, the conjugate linker does not comprise an amide.In certain embodiments, the conjugate linker comprises a polyamide.

In certain embodiments, the conjugate linker comprises an amine.

In certain embodiments, the conjugate linker comprises one or moredisulfide bonds.

In certain embodiments, the conjugate linker comprises a protein bindingmoiety. In certain embodiments, the protein binding moiety comprises alipid. In certain embodiments, the protein binding moiety is selectedfrom among: cholesterol, cholic acid, adamantane acetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol,geranyloxyhexyl group, hexadecylglycerol, borneol, menthol,1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl,or phenoxazine), a vitamin (e.g., folate, vitamin A, vitamin E, biotin,pyridoxal), a peptide, a carbohydrate (e.g., monosaccharide,disaccharide, trisaccharide, tetrasaccharide, oligosaccharide,polysaccharide), an endosomolytic component, a steroid (e.g., uvaol,hecigenin, diosgenin), a terpene (e.g., triterpene, e.g.,sarsasapogenin, friedelin, epifriedelanol derivatized lithocholic acid),or a cationic lipid. In certain embodiments, the protein binding moietyis selected from among: a C16 to C22 long chain saturated or unsaturatedfatty acid, cholesterol, cholic acid, vitamin E, adamantane or1-pentafluoropropyl.

In certain embodiments, the conjugate linker has a structure selectedfrom among:

wherein each n is, independently, is from 1 to 20; and p is from 1 to 6.

In certain embodiments, the conjugate linker has a structure selectedfrom among:

wherein each n is, independently, from 1 to 20.

In certain embodiments, the conjugate linker has a structure selectedfrom among:

In certain embodiments, the conjugate linker has a structure selectedfrom among:

wherein n is from 1 to 20.

In certain embodiments, the conjugate linker has a structure selectedfrom among:

In certain embodiments, the conjugate linker has a structure selectedfrom among:

wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.

In certain embodiments, the conjugate linker has the followingstructure:

In certain embodiments, the branching group has one of the followingstructures:

wherein each A₁ is independently, O, S, C═O or NH; and

each n is, independently, from 1 to 20.

In certain embodiments, the branching group has one of the followingstructures:

-   -   wherein each A₁ is independently, O, S, C═O or NH; and    -   each n is, independently, from 1 to 20.

In certain embodiments, the branching group has the following structure:

In certain embodiments, the branching group has the following structure:

In certain embodiments, the branching group has the following structure:

In certain embodiments, the branching group has the following structure:

In certain embodiments, the branching group comprises an ether.

In certain embodiments, the branching group has the following structure:

each n is, independently, from 1 to 20; and

m is from 2 to 6.

In certain embodiments, the branching group has the following structure:

In certain embodiments, the branching group has the following structure:

In certain embodiments, the branching group comprises:

wherein each j is an integer from 1 to 3; and

wherein each n is an integer from 1 to 20.

In certain embodiments, the branching group comprises:

In certain embodiments, each tether is selected from among:

wherein L is selected from a phosphorus linking group and a neutrallinking group;

-   -   Z₁ is C(═O)O—R₂;    -   Z₂ is H, C₁-C₆ alkyl or substituted C₁-C₆ alky;    -   R₂ is H, C₁-C₆ alkyl or substituted C₁-C₆ alky; and    -   each m₁ is, independently, from 0 to 20 wherein at least one m₁        is greater than 0 for each tether.

In certain embodiments, each tether is selected from among:

wherein Z₂ is H or CH₃; and

each m₂ is, independently, from 0 to 20 wherein at least one m₂ isgreater than 0 for each tether.

In certain embodiments, each tether is selected from among:

wherein n is from 1 to 12; and

wherein m is from 1 to 12.

In certain embodiments, at least one tether comprises ethylene glycol.

In certain embodiments, at least one tether comprises an amide. Incertain embodiments, at least one tether comprises a polyamide.

In certain embodiments, at least one tether comprises an amine.

In certain embodiments, at least two tethers are different from oneanother. In certain embodiments, all of the tethers are the same as oneanother.

In certain embodiments, each tether is selected from among:

wherein each n is, independently, from 1 to 20; and

each p is from 1 to about 6.

In certain embodiments, each tether is selected from among:

In certain embodiments, each tether has the following structure:

wherein each n is, independently, from 1 to 20.

In certain embodiments, each tether has the following structure:

In certain embodiments, the tether has a structure selected from among:

wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.

In certain embodiments, the tether has a structure selected from among:

In certain embodiments, the ligand is galactose.

In certain embodiments, the ligand is mannose-6-phosphate.

In certain embodiments, each ligand is selected from among:

wherein each R₁ is selected from OH and NHCOOH.

In certain embodiments, each ligand is selected from among:

In certain embodiments, each ligand has the following structure:

In certain embodiments, each ligand has the following structure:

In certain embodiments, the conjugate group comprises a cell-targetingmoiety.

In certain embodiments, the conjugate group comprises a cell-targetingmoiety having the following structure:

wherein each n is, independently, from 1 to 20.

In certain embodiments, the cell-targeting moiety has the followingstructure:

In certain embodiments, the cell-targeting moiety has the followingstructure:

wherein each n is, independently, from 1 to 20.

In certain embodiments, the cell-targeting moiety has the followingstructure:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety has the followingstructure:

In certain embodiments, the cell-targeting moiety has the followingstructure:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety has the followingstructure

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety has the followingstructure:

In certain embodiments, the cell-targeting moiety has the followingstructure:

In certain embodiments, the cell-targeting moiety has the followingstructure:

In certain embodiments, the cell-targeting moiety has the followingstructure:

In certain embodiments, the cell-targeting moiety has the followingstructure:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety has the followingstructure:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety has the followingstructure:

In certain embodiments, the cell-targeting moiety comprises:

wherein each Y is selected from O, S, a substituted or unsubstitutedC₁-C₁₀ alkyl, amino, substituted amino, azido, alkenyl or alkynyl.

In certain embodiments, the conjugate group comprises:

wherein each Y is selected from O, S, a substituted or unsubstitutedC₁-C₁₀ alkyl, amino, substituted amino, azido, alkenyl or alkynyl.

In certain embodiments, the cell-targeting moiety has the followingstructure:

wherein each Y is selected from O, S, a substituted or unsubstitutedC₁-C₁₀ alkyl, amino, substituted amino, azido, alkenyl or alkynyl.

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises a cleavable moietyselected from among: a phosphodiester, an amide, or an ester.

In certain embodiments, the conjugate group comprises a phosphodiestercleavable moiety.

In certain embodiments, the conjugate group does not comprise acleavable moiety, and wherein the conjugate group comprises aphosphorothioate linkage between the conjugate group and theoligonucleotide.

In certain embodiments, the conjugate group comprises an amide cleavablemoiety.

In certain embodiments, the conjugate group comprises an ester cleavablemoiety.

In certain embodiments, the compound has the following structure:

wherein each n is, independently, from 1 to 20;

Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein each n is, independently, from 1 to 20;

Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein each n is, independently, from 1 to 20;

Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide;

Z is H or a linked solid support; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein each n is, independently, from 1 to 20;

Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide;

Z is H or a linked solid support; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the conjugate group comprises:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the conjugate group comprises:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the conjugate group comprises:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, B_(x) is selected from among from adenine,guanine, thymine, uracil, or cytosine, or 5-methyl cytosine. In certainembodiments, B_(x) is adenine. In certain embodiments, BX is thymine. Incertain embodiments, Q₁₃ is O(CH₂)₂—OCH₃. In certain embodiments, Q₁₃ isH.

Certain embodiments of the invention provide a prodrug comprising thecompositions or compounds disclosed herein. Certain embodiments providemethods of using the conjugated antisense compounds and compositionsdescribed herein for inhibiting ANGPTL3 expression. In certainembodiments, the conjugated antisense compounds or compositions inhibitANGPTL3 by at least 5%, at least 10%, at least 20%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90% or at least 95%. In a preferred embodiment, theantisense compound comprising a modified oligonucleotide and a conjugategroup decreases ANGPTL3 by at least 50%. In a preferred embodiment, theantisense compound comprising a modified oligonucleotide and a conjugategroup decreases ANGPTL3 by at least 55%. In a preferred embodiment theantisense compound comprising a modified oligonucleotide and a conjugategroup decreases ANGPTL3 by at least 60%. In a preferred embodiment, theantisense compound comprising a modified oligonucleotide and a conjugategroup decreases ANGPTL3 by at least 65%. In a preferred embodiment, theantisense compound comprising a modified oligonucleotide and a conjugategroup decreases ANGPTL3 by at least 70%. In a preferred embodiment, theantisense compound comprising a modified oligonucleotide and a conjugategroup decreases ANGPTL3 by at least 75%. In a preferred embodiment, theantisense compound comprising a modified oligonucleotide and a conjugategroup decreases ANGPTL3 by at least 80%. In a preferred embodiment, theantisense compound comprising a modified oligonucleotide and a conjugategroup decreases ANGPTL3 by at least 85%. In a preferred embodiment, theantisense compound comprising a modified oligonucleotide and a conjugategroup decreases ANGPTL3 by at least 90%. In a preferred embodiment, theantisense compound comprising a modified oligonucleotide and a conjugategroup decreases ANGPTL3 by at least 95%.

In certain embodiments, the conjugated antisense compounds orcompositions disclosed herein have an IC₅₀ of less than 20 μM, less than10 μM, less than 8 μM, less than 5 μM, less than 2 μM, less than 1 μM,or less than 0.8 μM, when tested human cells, for example, in the Hep3Bcell line as described in Examples 2-3 and 7-10.

In certain embodiments, the conjugated antisense compounds orcompositions disclosed herein are efficacious by virtue of having aviscosity of less than 40 cP, less than 35 cP, less than 30 cP, lessthan 25 cP, less than 20 cP or less than 15 cP when measured by theparameters as described in Example 13.

In certain embodiments, the conjugated antisense compounds orcompositions disclosed herein are highly tolerable, as demonstrated bythe in vivo tolerability measurements described in the examples. Incertain embodiments, the conjugated antisense compounds as describedherein are highly tolerable, as demonstrated by having an increase inALT and/or AST value of no more than 4 fold, 3 fold, 2 fold or 1.5 foldover saline treated animals.

Certain embodiments disclosed herein provide a salt of the conjugatedantisense compounds disclosed herein. In certain embodiments, thecompounds or compositions disclosed herein comprise a salt of themodified oligonucleotide with the conjugate group.

In certain embodiments, the conjugated antisense compounds orcompositions disclosed herein further comprise a pharmaceuticallyacceptable carrier or diluent.

In certain embodiments, the animal is a human.

Certain embodiments disclosed herein provide methods comprisingadministering to an animal the conjugated antisense compounds orcompositions disclosed herein. In certain embodiments, administering theconjugated antisense compound or composition is therapeutic. In certainembodiments, administering the conjugated antisense compound orcomposition treats, prevents, or slows progression of a disease relatedto ANGPTL3. In certain embodiments, the disease is related to elevatedANGPTL3. In certain embodiments, administering the conjugated antisensecompound or composition prevents, treats, ameliorates, or slowsprogression of a cardiovascular and/or metabolic disease.

Certain embodiments disclosed herein provide methods for treating ahuman with a cardiovascular and/or metabolic disease comprisingidentifying a human with cardiovascular and/or metabolic disease andadministering to the human a therapeutically effective amount of any ofthe conjugated antisense compounds or compositions disclosed herein, soas to treat the human for cardiovascular and/or metabolic disease.

Certain embodiments provide conjugated antisense compounds andcompositions described herein for use in therapy. In certainembodiments, the therapy is used in treating, preventing, or slowingprogression of a disease related to ANGPTL3. In certain embodiments, thetherapy is used in treating, preventing, or slowing progression of adisease related to elevated ANGPTL3.

In certain embodiments, the disease is a cardiovascular and/or metabolicdisease, disorder or condition. In certain embodiments, the metabolicand/or cardiovascular disease includes, but is not limited to, obesity,diabetes, insulin resistance, atherosclerosis, dyslipidemia,lipodystrophy, coronary heart disease, non-alcoholic fatty liver disease(NAFLD), nonalcoholic steatohepatitis (NASH) hyperfattyacidemia ormetabolic syndrome, or a combination thereof. The dyslipidemia can behyperlipidemia. The hyperlipidemia can be combined hyperlipidemia (CHL),hypercholesterolemia, hypertriglyceridemia, or both hypercholesterolemiaand hypertriglyceridemia. The combined hyperlipidemia can be familial ornon-familial. The hypercholesterolemia can be familial homozygoushypercholesterolemia (HoFH), familial heterozygous hypercholesterolemia(HeFH). The hypertriglyceridemia can be familial chylomicronemiasyndrome (FCS) or hyperlipoproteinemia Type IV. The NAFLD can be hepaticsteatosis or steatohepatitis. The diabetes can be type 2 diabetes ortype 2 diabetes with dyslipidemia. The insulin resistance can be insulinresistance with dyslipidemia.

In certain embodiments, the conjugated antisense compounds orcompositions disclosed herein are designated as a first agent and themethods or uses disclosed herein further comprise administering a secondagent. In certain embodiments, the first agent and the second agent areco-administered. In certain embodiments the first agent and the secondagent are co-administered sequentially or concomitantly.

In certain embodiments, the second agent is a glucose-lowering agent.The glucose lowering agent can include, but is not limited to, atherapeutic lifestyle change, PPAR agonist, a dipeptidyl peptidase (IV)inhibitor, a GLP-1 analog, insulin or an insulin analog, an insulinsecretagogue, a SGLT2 inhibitor, a human amylin analog, a biguanide, analpha-glucosidase inhibitor, or a combination thereof. Theglucose-lowering agent can include, but is not limited to metformin,sulfonylurea, rosiglitazone, meglitinide, thiazolidinedione,alpha-glucosidase inhibitor or a combination thereof. The sulfonylureacan be acetohexamide, chlorpropamide, tolbutamide, tolazamide,glimepiride, a glipizide, a glyburide, or a gliclazide. The meglitinidecan be nateglinide or repaglinide. The thiazolidinedione can bepioglitazone or rosiglitazone. The alpha-glucosidase can be acarbose ormiglitol.

In certain embodiments, the second agent is a lipid-lowering therapy. Incertain embodiments the lipid lowering therapy can include, but is notlimited to, a therapeutic lifestyle change, HMG-CoA reductase inhibitor,cholesterol absorption inhibitor, MTP inhibitor (e.g., a small molecule,polypeptide, antibody or antisense compound targeted to MTP), ApoBinhibitor (e.g., a small molecule, polypeptide, antibody or antisensecompound targeted to ApoB), ApoC3 inhibitor (e.g., a small molecule,polypeptide, antibody or antisense compound targeted to ApoC3), PCSK9inhibitor (e.g., a small molecule, polypeptide, antibody or antisensecompound targeted to PCSK9), CETP inhibitor (e.g., a small molecule,polypeptide, antibody or antisense compound targeted to CETP), fibrate,beneficial oil (e.g., krill or fish oils (e.g., Vascepa®), flaxseed oil,or other oils rich in omega-3 fatty acids such as α-linolenic acid(ALA), docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA)), orany combination thereof. The HMG-CoA reductase inhibitor can beatorvastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin, orsimvastatin. The cholesterol absorption inhibitor can be ezetimibe. Thefibrate can be fenofibrate, bezafibrate, ciprofibrate, clofibrate,gemfibrozil and the like.

In certain embodiments, administration comprises parenteraladministration. In certain embodiments, administration comprisessubcutaneous administration.

In certain embodiments, administering a conjugated antisense compounddisclosed herein results in a reduction of lipid levels, includingtriglyceride levels, cholesterol levels, insulin resistance, glucoselevels or a combination thereof. One or more of the levels can beindependently reduced by at least 5%, at least 10%, at least 20%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90% or at least 95%. Administering theconjugated antisense compound can result in improved insulin sensitivityor hepatic insulin sensitivity. Administering the conjugated antisensecompound disclosed herein can result in a reduction in atheroscleroticplaques, obesity, glucose, lipids, glucose resistance, cholesterol, orimprovement in insulin sensitivity or any combination thereof.

Certain embodiments provide the use of a conjugated antisense compoundas described herein in the manufacture of a medicament for treating,ameliorating, delaying or preventing one or more of a disease related toANGPTL3. Certain embodiments provide the use of a conjugated antisensecompound as described herein in the manufacture of a medicament fortreating, ameliorating, delaying or preventing one or more of ametabolic disease or a cardiovascular disease.

Certain embodiments provide a kit for treating, preventing, orameliorating one or more of a metabolic disease or a cardiovasculardisease as described herein wherein the kit comprises: a) a conjugatedantisense compound as described herein; and optionally b) an additionalagent or therapy as described herein. The kit can further includeinstructions or a label for using the kit to treat, prevent, orameliorate one or more of a metabolic disease or a cardiovasculardisease.

Antisense Compounds

Oligomeric compounds include, but are not limited to, oligonucleotides,oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics,antisense compounds, antisense oligonucleotides, and siRNAs. Anoligomeric compound can be “antisense” to a target nucleic acid, meaningthat is capable of undergoing hybridization to a target nucleic acidthrough hydrogen bonding.

In certain embodiments, an antisense compound has a nucleobase sequencethat, when written in the 5′ to 3′ direction, comprises the reversecomplement of the target segment of a target nucleic acid to which it istargeted. In certain such embodiments, an antisense oligonucleotide hasa nucleobase sequence that, when written in the 5′ to 3′ direction,comprises the reverse complement of the target segment of a targetnucleic acid to which it is targeted.

In certain embodiments, an antisense compound targeted to ANGPTL3nucleic acid is 10 to 30 nucleotides in length. In other words,antisense compounds are from 10 to 30 linked nucleobases. In otherembodiments, the antisense compound comprises a modified oligonucleotideconsisting of 8 to 80, 10 to 80, 12 to 50, 12 to 30, 15 to 30, 18 to 24,19 to 22, or 20 linked nucleobases. In certain such embodiments, theantisense compound comprises a modified oligonucleotide consisting of 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or80 linked nucleobases in length, or a range defined by any two of theabove values.

In certain embodiments, the antisense compound comprises a shortened ortruncated modified oligonucleotide. The shortened or truncated modifiedoligonucleotide can have a single nucleoside deleted from the 5′ end (5′truncation), or alternatively from the 3′ end (3′ truncation). Ashortened or truncated oligonucleotide can have two or more nucleosidesdeleted from the 5′ end, or alternatively can have two or morenucleosides deleted from the 3′ end. Alternatively, the deletednucleosides can be dispersed throughout the modified oligonucleotide,for example, in an antisense compound having one or more nucleosidedeleted from the 5′ end and one or more nucleoside deleted from the 3′end.

When a single additional nucleoside is present in a lengthenedoligonucleotide, the additional nucleoside can be located at the 5′, 3′end or central portion of the oligonucleotide. When two or moreadditional nucleosides are present, the added nucleosides can beadjacent to each other, for example, in an oligonucleotide having twonucleosides added to the 5′ end (5′ addition), or alternatively to the3′ end (3′ addition) or the central portion, of the oligonucleotide.Alternatively, the added nucleoside can be dispersed throughout theantisense compound, for example, in an oligonucleotide having one ormore nucleoside added to the 5′ end, one or more nucleoside added to the3′ end, and/or one or more nucleoside added to the central portion.

It is possible to increase or decrease the length of an antisensecompound, such as an antisense oligonucleotide, and/or introducemismatch bases without eliminating activity. For example, in Woolf etal. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series ofantisense oligonucleotides 13-25 nucleobases in length were tested fortheir ability to induce cleavage of a target RNA in an oocyte injectionmodel. Antisense oligonucleotides 25 nucleobases in length with 8 or 11mismatch bases near the ends of the antisense oligonucleotides were ableto direct specific cleavage of the target mRNA, albeit to a lesserextent than the antisense oligonucleotides that contained no mismatches.Similarly, target specific cleavage was achieved using 13 nucleobaseantisense oligonucleotides, including those with 1 or 3 mismatches.

Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March 2001)demonstrated the ability of an oligonucleotide having 100%complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xLmRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and invivo. Furthermore, this oligonucleotide demonstrated potent anti-tumoractivity in vivo.

Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a seriesof tandem 14 nucleobase antisense oligonucleotides, and a 28 and 42nucleobase antisense oligonucleotides comprised of the sequence of twoor three of the tandem antisense oligonucleotides, respectively, fortheir ability to arrest translation of human DHFR in a rabbitreticulocyte assay. Each of the three 14 nucleobase antisenseoligonucleotides alone was able to inhibit translation, albeit at a moremodest level than the 28 or 42 nucleobase antisense oligonucleotides.

Certain Antisense Compound Motifs and Mechanisms

In certain embodiments, antisense compounds have chemically modifiedsubunits arranged in patterns, or motifs, to confer to the antisensecompounds properties such as enhanced inhibitory activity, increasedbinding affinity for a target nucleic acid, or resistance to degradationby in vivo nucleases. Chimeric antisense compounds typically contain atleast one region modified so as to confer increased resistance tonuclease degradation, increased cellular uptake, increased bindingaffinity for the target nucleic acid, and/or increased inhibitoryactivity. A second region of a chimeric antisense compound may conferanother desired property e.g., serve as a substrate for the cellularendonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.

Antisense activity may result from any mechanism involving thehybridization of the antisense compound (e.g., oligonucleotide) with atarget nucleic acid, wherein the hybridization ultimately results in abiological effect. In certain embodiments, the amount and/or activity ofthe target nucleic acid is modulated. In certain embodiments, the amountand/or activity of the target nucleic acid is reduced. In certainembodiments, hybridization of the antisense compound to the targetnucleic acid ultimately results in target nucleic acid degradation. Incertain embodiments, hybridization of the antisense compound to thetarget nucleic acid does not result in target nucleic acid degradation.In certain such embodiments, the presence of the antisense compoundhybridized with the target nucleic acid (occupancy) results in amodulation of antisense activity. In certain embodiments, antisensecompounds having a particular chemical motif or pattern of chemicalmodifications are particularly suited to exploit one or more mechanisms.In certain embodiments, antisense compounds function through more thanone mechanism and/or through mechanisms that have not been elucidated.Accordingly, the antisense compounds described herein are not limited byparticular mechanism.

Antisense mechanisms include, without limitation, RNase H mediatedantisense; RNAi mechanisms, which utilize the RISC pathway and include,without limitation, siRNA, ssRNA and microRNA mechanisms; and occupancybased mechanisms. Certain antisense compounds may act through more thanone such mechanism and/or through additional mechanisms.

RNase H-Mediated Antisense

In certain embodiments, antisense activity results at least in part fromdegradation of target RNA by RNase H. RNase H is a cellular endonucleasethat cleaves the RNA strand of an RNA:DNA duplex. It is known in the artthat single-stranded antisense compounds which are “DNA-like” elicitRNase H activity in mammalian cells. Accordingly, antisense compoundscomprising at least a portion of DNA or DNA-like nucleosides mayactivate RNase H, resulting in cleavage of the target nucleic acid. Incertain embodiments, antisense compounds that utilize RNase H compriseone or more modified nucleosides. In certain embodiments, such antisensecompounds comprise at least one block of 1-8 modified nucleosides. Incertain such embodiments, the modified nucleosides do not support RNaseH activity. In certain embodiments, such antisense compounds aregapmers, as described herein. In certain such embodiments, the gap ofthe gapmer comprises DNA nucleosides. In certain such embodiments, thegap of the gapmer comprises DNA-like nucleosides. In certain suchembodiments, the gap of the gapmer comprises DNA nucleosides andDNA-like nucleosides.

Certain antisense compounds having a gapmer motif are consideredchimeric antisense compounds. In a gapmer an internal region having aplurality of nucleotides that supports RNaseH cleavage is positionedbetween external regions having a plurality of nucleotides that arechemically distinct from the nucleosides of the internal region. In thecase of an antisense oligonucleotide having a gapmer motif, the gapsegment generally serves as the substrate for endonuclease cleavage,while the wing segments comprise modified nucleosides. In certainembodiments, the regions of a gapmer are differentiated by the types ofsugar moieties comprising each distinct region. The types of sugarmoieties that are used to differentiate the regions of a gapmer may insome embodiments include β-D-ribonucleosides, β-D-deoxyribonucleosides,2′-modified nucleosides (such 2′-modified nucleosides may include 2′-MOEand 2′-O—CH₃, among others), and bicyclic sugar modified nucleosides(such bicyclic sugar modified nucleosides may include those having aconstrained ethyl). In certain embodiments, nucleosides in the wings mayinclude several modified sugar moieties, including, for example 2′-MOEand bicyclic sugar moieties such as constrained ethyl or LNA. In certainembodiments, wings may include several modified and unmodified sugarmoieties. In certain embodiments, wings may include various combinationsof 2′-MOE nucleosides, bicyclic sugar moieties such as constrained ethylnucleosides or LNA nucleosides, and 2′-deoxynucleosides.

Each distinct region may comprise uniform sugar moieties, variant, oralternating sugar moieties. The wing-gap-wing motif is frequentlydescribed as “X—Y—Z”, where “X” represents the length of the 5′-wing,“Y” represents the length of the gap, and “Z” represents the length ofthe 3′-wing. “X” and “Z” may comprise uniform, variant, or alternatingsugar moieties. In certain embodiments, “X” and “Y” may include one ormore 2′-deoxynucleosides. “Y” may comprise 2′-deoxynucleosides. As usedherein, a gapmer described as “X—Y—Z” has a configuration such that thegap is positioned immediately adjacent to each of the 5′-wing and the 3′wing. Thus, no intervening nucleotides exist between the 5′-wing andgap, or the gap and the 3′-wing. Any of the antisense compoundsdescribed herein can have a gapmer motif. In certain embodiments, “X”and “Z” are the same; in other embodiments they are different. Incertain embodiments, “Y” is between 8 and 15 nucleosides. X, Y, or Z canbe any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 25, 30 or more nucleosides.

In certain embodiments, the antisense compound targeted to an ANGPTL3nucleic acid has a gapmer motif in which the gap consists of 6, 7, 8, 9,10, 11, 12, 13, 14, 15, or 16 linked nucleosides.

In certain embodiments, the antisense oligonucleotide has a sugar motifdescribed by Formula A as follows:(J)_(m)-(B)_(n)-(J)_(p)-(B)_(r)-(A)_(t)-(D)_(g)-(A)_(v)-(B)_(w)-(J)_(x)-(B)_(y)-(J)_(z)

wherein:

each A is independently a 2′-substituted nucleoside;

each B is independently a bicyclic nucleoside;

each J is independently either a 2′-substituted nucleoside or a2′-deoxynucleoside;

each D is a 2′-deoxynucleoside;

m is 0-4; n is 0-2; p is 0-2; r is 0-2; t is 0-2; v is 0-2; w is 0-4; xis 0-2; y is 0-2; z is 0-4; g is 6-14; provided that:

-   -   at least one of m, n, and r is other than 0;    -   at least one of w and y is other than 0;    -   the sum of m, n, p, r, and t is from 2 to 5; and the sum of v,        w, x, y, and z is from 2 to 5.

RNAi Compounds

In certain embodiments, antisense compounds are interfering RNAcompounds (RNAi), which include double-stranded RNA compounds (alsoreferred to as short-interfering RNA or siRNA) and single-stranded RNAicompounds (or ssRNA). Such compounds work at least in part through theRISC pathway to degrade and/or sequester a target nucleic acid (thus,include microRNA/microRNA-mimic compounds). In certain embodiments,antisense compounds comprise modifications that make them particularlysuited for such mechanisms.

1. ssRNA Compounds

In certain embodiments, antisense compounds including those particularlysuited for use as single-stranded RNAi compounds (ssRNA) comprise amodified 5′-terminal end. In certain such embodiments, the 5′-terminalend comprises a modified phosphate moiety. In certain embodiments, suchmodified phosphate is stabilized (e.g., resistant todegradation/cleavage compared to unmodified 5′-phosphate). In certainembodiments, such 5′-terminal nucleosides stabilize the 5′-phosphorousmoiety. Certain modified 5′-terminal nucleosides may be found in theart, for example in WO/2011/139702.

In certain embodiments, the 5′-nucleoside of an ssRNA compound hasFormula IIc:

wherein:

T₁ is an optionally protected phosphorus moiety;

T₂ is an internucleoside linking group linking the compound of FormulaIIc to the oligomeric compound;

A has one of the formulas:

Q₁ and Q₂ are each, independently, H, halogen, C₁-C₆ alkyl, substitutedC₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl orN(R₃)(R₄);

Q₃ is O, S, N(R₅) or C(R₆)(R₇);

each R₃, R₄ R₅, R₆ and R₇ is, independently, H, C₁-C₆ alkyl, substitutedC₁-C₆ alkyl or C₁-C₆ alkoxy;

M₃ is O, S, NR₁₄, C(R₁₅)(R₁₆), C(R₁₅)(R₁₆)C(R₁₇)(R₁₈), C(R₁₅)═C(R₁₇),OC(R₁₅)(R₁₆) or OC(R₁₅)(Bx₂);

R₁₄ is H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy,substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl,C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

R₁₅, R₁₆, R₁₇ and R₁₈ are each, independently, H, halogen, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆alkynyl;

Bx₁ is a heterocyclic base moiety;

or if Bx₂ is present then Bx₂ is a heterocyclic base moiety and Bx₁ isH, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy,substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl,C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

J₄, J₅, J₆ and J₇ are each, independently, H, halogen, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆alkynyl;

or J₄ forms a bridge with one of J₅ or J₇ wherein said bridge comprisesfrom 1 to 3 linked biradical groups selected from O, S, NR₁₉,C(R₂₀)(R₂₁), C(R₂₀)═C(R₂₁), C[═C(R₂₀)(R₂₁)] and C(═O) and the other twoof J₅, J₆ and J₇ are each, independently, H, halogen, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆alkynyl;

each R₁₉, R₂₀ and R₂₁ is, independently, H, C₁-C₆ alkyl, substitutedC₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

G is H, OH, halogen or O—[C(R₈)(R₉)]_(n)—[(C═O)_(m)—X₁]_(j)—Z;

each R₈ and R₉ is, independently, H, halogen, C₁-C₆ alkyl or substitutedC₁-C₆ alkyl;

X₁ is O, S or N(E₁);

Z is H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl orN(E₂)(E₃);

E₁, E₂ and E₃ are each, independently, H, C₁-C₆ alkyl or substitutedC₁-C₆ alkyl;

n is from 1 to about 6;

m is 0 or 1;

j is 0 or 1;

each substituted group comprises one or more optionally protectedsubstituent groups independently selected from halogen, OJ₁, N(J₁)(J₂),=NJ₁, SJ₁, N₃, CN, OC(═X₂)J₁, OC(═X₂)N(J₁)(J₂) and C(═X₂)N(J₁)(J₂);

X₂ is O, S or NJ₃;

each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl;

when j is 1 then Z is other than halogen or N(E₂)(E₃); and

wherein said oligomeric compound comprises from 8 to 40 monomericsubunits and is hybridizable to at least a portion of a target nucleicacid.

In certain embodiments, M₃ is O, CH═CH, OCH₂ or OC(H)(Bx₂). In certainembodiments, M₃ is O.

In certain embodiments, J₄, J₅, J₆ and J₇ are each H. In certainembodiments, J₄ forms a bridge with one of J₅ or J₇.

In certain embodiments, A has one of the formulas:

wherein:

Q₁ and Q₂ are each, independently, H, halogen, C₁-C₆ alkyl, substitutedC₁-C₆ alkyl, C₁-C₆ alkoxy or substituted C₁-C₆ alkoxy. In certainembodiments, Q₁ and Q₂ are each H. In certain embodiments, Q₁ and Q₂ areeach, independently, H or halogen. In certain embodiments, Q₁ and Q₂ isH and the other of Q₁ and Q₂ is F, CH₃ or OCH₃.

In certain embodiments, T₁ has the formula:

wherein:

R_(a) and R_(c) are each, independently, protected hydroxyl, protectedthiol, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substitutedC₁-C₆ alkoxy, protected amino or substituted amino; and

R_(b) is O or S. In certain embodiments, R_(b) is O and R_(a) and R, areeach, independently, OCH₃, OCH₂CH₃ or CH(CH₃)₂.

In certain embodiments, G is halogen, OCH₃, OCH₂F, OCHF₂, OCF₃, OCH₂CH₃,O(CH₂)₂F, OCH₂CHF₂, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—SCH₃,O(CH₂)₂—OCF₃, O(CH₂)₃—N(R₁₀)(R₁₁), O(CH₂)₂—ON(R₁₀)(R₁₁),O(CH₂)₂—O(CH₂)₂—N(R₁₀)(R₁), OCH₂C(═O)—N(R₁₀)(R₁),OCH₂C(═O)—N(R₁₂)—(CH₂)₂—N(R₁₀)(R₁) orO(CH₂)₂—N(R₁₂)—C(═NR₁₃)[N(R₁₀)(R₁₁)] wherein R₁₀, R₁₁, R₁₂ and R₁₃ areeach, independently, H or C₁-C₆ alkyl. In certain embodiments, G ishalogen, OCH₃, OCF₃, OCH₂CH₃, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃,O(CH₂)₂—O(CH₂)₂—N(CH₃)₂, OCH₂C(═O)—N(H)CH₃,OCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂ or OCH₂—N(H)—C(═NH)NH₂. In certainembodiments, G is F, OCH₃ or O(CH₂)₂—OCH₃. In certain embodiments, G isO(CH₂)₂—OCH₃.

In certain embodiments, the 5′-terminal nucleoside has Formula IIe:

In certain embodiments, antisense compounds, including thoseparticularly suitable for ssRNA comprise one or more type of modifiedsugar moieties and/or naturally occurring sugar moieties arranged alongan oligonucleotide or region thereof in a defined pattern or sugarmodification motif. Such motifs may include any of the sugarmodifications discussed herein and/or other known sugar modifications.

In certain embodiments, the oligonucleotides comprise or consist of aregion having uniform sugar modifications. In certain such embodiments,each nucleoside of the region comprises the same RNA-like sugarmodification. In certain embodiments, each nucleoside of the region is a2′-F nucleoside. In certain embodiments, each nucleoside of the regionis a 2′-OMe nucleoside. In certain embodiments, each nucleoside of theregion is a 2′-MOE nucleoside. In certain embodiments, each nucleosideof the region is a cEt nucleoside. In certain embodiments, eachnucleoside of the region is an LNA nucleoside. In certain embodiments,the uniform region constitutes all or essentially all of theoligonucleotide. In certain embodiments, the region constitutes theentire oligonucleotide except for 1-4 terminal nucleosides.

In certain embodiments, oligonucleotides comprise one or more regions ofalternating sugar modifications, wherein the nucleosides alternatebetween nucleotides having a sugar modification of a first type andnucleotides having a sugar modification of a second type. In certainembodiments, nucleosides of both types are RNA-like nucleosides. Incertain embodiments the alternating nucleosides are selected from:2′-OMe, 2′-F, 2′-MOE, LNA, and cEt. In certain embodiments, thealternating modificatios are 2′-F and 2′-OMe. Such regions may becontiguous or may be interupted by differently modified nucleosides orconjugated nucleosides.

In certain embodiments, the alternating region of alternatingmodifications each consist of a single nucleoside (i.e., the patern is(AB)_(x)A_(y) wheren A is a nucleoside having a sugar modification of afirst type and B is a nucleoside having a sugar modification of a secondtype; x is 1-20 and y is 0 or 1). In certan embodiments, one or morealternating regions in an alternating motif includes more than a singlenucleoside of a type. For example, oligonucleotides may include one ormore regions of any of the following nucleoside motifs:

AABBAA; ABBABB; AABAAB; ABBABAABB; ABABAA; AABABAB; ABABAA;ABBAABBABABAA; BABBAABBABABAA; or ABABBAABBABABAA;

wherein A is a nucleoside of a first type and B is a nucleoside of asecond type. In certain embodiments, A and B are each selected from2′-F, 2′-OMe, BNA, and MOE.

In certain embodiments, oligonucleotides having such an alternatingmotif also comprise a modified 5′ terminal nucleoside, such as those offormula IIc or lie.

In certain embodiments, oligonucleotides comprise a region having a2-2-3 motif. Such regions comprises the following motif:

-(A)₂-(B)_(x)-(A)₂-(C)_(y)-(A)₃-

wherein: A is a first type of modified nucleoside;

B and C, are nucleosides that are differently modified than A, however,B and C may have the same or different modifications as one another;

x and y are from 1 to 15.

In certain embodiments, A is a 2′-OMe modified nucleoside. In certainembodiments, B and C are both 2′-F modified nucleosides. In certainembodiments, A is a 2′-OMe modified nucleoside and B and C are both 2′-Fmodified nucleosides.

In certain embodiments, oligonucleosides have the following sugar motif:

5′-(Q)-(AB)_(x)A_(y)-(D)_(z)

wherein:

Q is a nucleoside comprising a stabilized phosphate moiety. In certainembodiments, Q is a nucleoside having Formula IIc or IIe;

A is a first type of modified nucleoside;

B is a second type of modified nucleoside;

D is a modified nucleoside comprising a modification different from thenucleoside adjacent to it. Thus, if y is 0, then D must be differentlymodified than B and if y is 1, then D must be differently modified thanA. In certain embodiments, D differs from both A and B.

X is 5-15;

Y is 0 or 1;

Z is 0-4.

In certain embodiments, oligonucleosides have the following sugar motif:

5′-(Q)-(A)_(x)-(D)_(z)

wherein:

Q is a nucleoside comprising a stabilized phosphate moiety. In certainembodiments, Q is a nucleoside having Formula IIc or IIe;

A is a first type of modified nucleoside;

D is a modified nucleoside comprising a modification different from A.

X is 11-30;

Z is 0-4.

In certain embodiments A, B, C, and D in the above motifs are selectedfrom: 2′-OMe, 2′-F, 2′-MOE, LNA, and cEt. In certain embodiments, Drepresents terminal nucleosides. In certain embodiments, such terminalnucleosides are not designed to hybridize to the target nucleic acid(though one or more might hybridize by chance). In certiain embodiments,the nucleobase of each D nucleoside is adenine, regardless of theidentity of the nucleobase at the corresponding position of the targetnucleic acid. In certain embodiments the nucleobase of each D nucleosideis thymine.

In certain embodiments, antisense compounds, including thoseparticularly suited for use as ssRNA comprise modified internucleosidelinkages arranged along the oligonucleotide or region thereof in adefined pattern or modified internucleoside linkage motif. In certainembodiments, oligonucleotides comprise a region having an alternatinginternucleoside linkage motif. In certain embodiments, oligonucleotidescomprise a region of uniformly modified internucleoside linkages. Incertain such embodiments, the oligonucleotide comprises a region that isuniformly linked by phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide is uniformly linked byphosphorothioate internucleoside linkages. In certain embodiments, eachinternucleoside linkage of the oligonucleotide is selected fromphosphodiester and phosphorothioate. In certain embodiments, eachinternucleoside linkage of the oligonucleotide is selected fromphosphodiester and phosphorothioate and at least one internucleosidelinkage is phosphorothioate.

In certain embodiments, the oligonucleotide comprises at least 6phosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide comprises at least 8 phosphorothioate internucleosidelinkages. In certain embodiments, the oligonucleotide comprises at least10 phosphorothioate internucleoside linkages. In certain embodiments,the oligonucleotide comprises at least one block of at least 6consecutive phosphorothioate internucleoside linkages. In certainembodiments, the oligonucleotide comprises at least one block of atleast 8 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least one block ofat least 10 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least one block ofat least one 12 consecutive phosphorothioate internucleoside linkages.In certain such embodiments, at least one such block is located at the3′ end of the oligonucleotide. In certain such embodiments, at least onesuch block is located within 3 nucleosides of the 3′ end of theoligonucleotide.

Oligonucleotides having any of the various sugar motifs describedherein, may have any linkage motif. For example, the oligonucleotides,including but not limited to those described above, may have a linkagemotif selected from non-limiting the table below:

5′ most linkage Central region 3′-region PS Alternating PO/PS 6 PS PSAlternating PO/PS 7 PS PS Alternating PO/PS 8 PS

2. siRNA Compounds

In certain embodiments, antisense compounds are double-stranded RNAicompounds (siRNA). In such embodiments, one or both strands may compriseany modification motif described above for ssRNA. In certainembodiments, ssRNA compounds may be unmodified RNA. In certainembodiments, siRNA compounds may comprise unmodified RNA nucleosides,but modified internucleoside linkages.

Several embodiments relate to double-stranded compositions wherein eachstrand comprises a motif defined by the location of one or more modifiedor unmodified nucleosides. In certain embodiments, compositions areprovided comprising a first and a second oligomeric compound that arefully or at least partially hybridized to form a duplex region andfurther comprising a region that is complementary to and hybridizes to anucleic acid target. It is suitable that such a composition comprise afirst oligomeric compound that is an antisense strand having full orpartial complementarity to a nucleic acid target and a second oligomericcompound that is a sense strand having one or more regions ofcomplementarity to and forming at least one duplex region with the firstoligomeric compound.

The compositions of several embodiments modulate gene expression byhybridizing to a nucleic acid target resulting in loss of its normalfunction. In some embodiments, the target nucleic acid is ANGPTL3. Incertain embodiment, the degradation of the targeted ANGPTL3 isfacilitated by an activated RISC complex that is formed withcompositions disclosed herein.

Several embodiments are directed to double-stranded compositions whereinone of the strands is useful in, for example, influencing thepreferential loading of the opposite strand into the RISC (or cleavage)complex.

The compositions are useful for targeting selected nucleic acidmolecules and modulating the expression of one or more genes. In someembodiments, the compositions of the present invention hybridize to aportion of a target RNA resulting in loss of normal function of thetarget RNA.

Certain embodiments are drawn to double-stranded compositions whereinboth the strands comprises a hemimer motif, a fully modified motif, apositionally modified motif or an alternating motif. Each strand of thecompositions of the present invention can be modified to fulfil aparticular role in for example the siRNA pathway. Using a differentmotif in each strand or the same motif with different chemicalmodifications in each strand permits targeting the antisense strand forthe RISC complex while inhibiting the incorporation of the sense strand.Within this model, each strand can be independently modified such thatit is enhanced for its particular role. The antisense strand can bemodified at the 5′-end to enhance its role in one region of the RISCwhile the 3′-end can be modified differentially to enhance its role in adifferent region of the RISC.

The double-stranded oligonucleotide molecules can be a double-strandedpolynucleotide molecule comprising self-complementary sense andantisense regions, wherein the antisense region comprises nucleotidesequence that is complementary to nucleotide sequence in a targetnucleic acid molecule or a portion thereof and the sense region havingnucleotide sequence corresponding to the target nucleic acid sequence ora portion thereof. The double-stranded oligonucleotide molecules can beassembled from two separate oligonucleotides, where one strand is thesense strand and the other is the antisense strand, wherein theantisense and sense strands are self-complementary (i.e. each strandcomprises nucleotide sequence that is complementary to nucleotidesequence in the other strand; such as where the antisense strand andsense strand form a duplex or double-stranded structure, for examplewherein the double-stranded region is about 15 to about 30, e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 basepairs; the antisense strand comprises nucleotide sequence that iscomplementary to nucleotide sequence in a target nucleic acid moleculeor a portion thereof and the sense strand comprises nucleotide sequencecorresponding to the target nucleic acid sequence or a portion thereof(e.g., about 15 to about 25 or more nucleotides of the double-strandedoligonucleotide molecule are complementary to the target nucleic acid ora portion thereof). Alternatively, the double-stranded oligonucleotideis assembled from a single oligonucleotide, where the self-complementarysense and antisense regions of the siRNA are linked by means of anucleic acid based or non-nucleic acid-based linker(s).

The double-stranded oligonucleotide can be a polynucleotide with aduplex, asymmetric duplex, hairpin or asymmetric hairpin secondarystructure, having self-complementary sense and antisense regions,wherein the antisense region comprises nucleotide sequence that iscomplementary to nucleotide sequence in a separate target nucleic acidmolecule or a portion thereof and the sense region having nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof. The double-stranded oligonucleotide can be a circularsingle-stranded polynucleotide having two or more loop structures and astem comprising self-complementary sense and antisense regions, whereinthe antisense region comprises nucleotide sequence that is complementaryto nucleotide sequence in a target nucleic acid molecule or a portionthereof and the sense region having nucleotide sequence corresponding tothe target nucleic acid sequence or a portion thereof, and wherein thecircular polynucleotide can be processed either in vivo or in vitro togenerate an active siRNA molecule capable of mediating RNAi.

In certain embodiments, the double-stranded oligonucleotide comprisesseparate sense and antisense sequences or regions, wherein the sense andantisense regions are covalently linked by nucleotide or non-nucleotidelinkers molecules as is known in the art, or are alternatelynon-covalently linked by ionic interactions, hydrogen bonding, van derwaals interactions, hydrophobic interactions, and/or stackinginteractions. In certain embodiments, the double-strandedoligonucleotide comprises nucleotide sequence that is complementary tonucleotide sequence of a target gene. In another embodiment, thedouble-stranded oligonucleotide interacts with nucleotide sequence of atarget gene in a manner that causes inhibition of expression of thetarget gene.

As used herein, double-stranded oligonucleotides need not be limited tothose molecules containing only RNA, but further encompasses chemicallymodified nucleotides and non-nucleotides. In certain embodiments, theshort interfering nucleic acid molecules lack 2′-hydroxy (2′-OH)containing nucleotides. In certain embodiments short interfering nucleicacids optionally do not include any ribonucleotides (e.g., nucleotideshaving a 2′-OH group). Such double-stranded oligonucleotides that do notrequire the presence of ribonucleotides within the molecule to supportRNAi can however have an attached linker or linkers or other attached orassociated groups, moieties, or chains containing one or morenucleotides with 2′-OH groups. Optionally, double-strandedoligonucleotides can comprise ribonucleotides at about 5, 10, 20, 30,40, or 50% of the nucleotide positions. As used herein, the term siRNAis meant to be equivalent to other terms used to describe nucleic acidmolecules that are capable of mediating sequence specific RNAi, forexample short interfering RNA (siRNA), double-stranded RNA (dsRNA),micro-RNA (miRNA), short hairpin RNA (shRNA), short interferingoligonucleotide, short interfering nucleic acid, short interferingmodified oligonucleotide, chemically modified siRNA,post-transcriptional gene silencing RNA (ptgsRNA), and others. Inaddition, as used herein, the term RNAi is meant to be equivalent toother terms used to describe sequence specific RNA interference, such aspost transcriptional gene silencing, translational inhibition, orepigenetics. For example, double-stranded oligonucleotides can be usedto epigenetically silence genes at both the post-transcriptional leveland the pre-transcriptional level. In a non-limiting example, epigeneticregulation of gene expression by siRNA molecules of the invention canresult from siRNA mediated modification of chromatin structure ormethylation pattern to alter gene expression (see, for example, Verdelet al., 2004, Science, 303, 672-676; Pal-Bhadra et al., 2004, Science,303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Volpe et al.,2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218;and Hall et al., 2002, Science, 297, 2232-2237).

It is contemplated that compounds and compositions of severalembodiments provided herein can target ANGPTL3 by a dsRNA-mediated genesilencing or RNAi mechanism, including, e.g., “hairpin” or stem-loopdouble-stranded RNA effector molecules in which a single RNA strand withself-complementary sequences is capable of assuming a double-strandedconformation, or duplex dsRNA effector molecules comprising two separatestrands of RNA. In various embodiments, the dsRNA consists entirely ofribonucleotides or consists of a mixture of ribonucleotides anddeoxynucleotides, such as the RNA/DNA hybrids disclosed, for example, byWO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filedApr. 21, 1999. The dsRNA or dsRNA effector molecule may be a singlemolecule with a region of self-complementarity such that nucleotides inone segment of the molecule base pair with nucleotides in anothersegment of the molecule. In various embodiments, a dsRNA that consistsof a single molecule consists entirely of ribonucleotides or includes aregion of ribonucleotides that is complementary to a region ofdeoxyribonucleotides. Alternatively, the dsRNA may include two differentstrands that have a region of complementarity to each other.

In various embodiments, both strands consist entirely ofribonucleotides, one strand consists entirely of ribonucleotides and onestrand consists entirely of deoxyribonucleotides, or one or both strandscontain a mixture of ribonucleotides and deoxyribonucleotides. Incertain embodiments, the regions of complementarity are at least 70, 80,90, 95, 98, or 100% complementary to each other and to a target nucleicacid sequence. In certain embodiments, the region of the dsRNA that ispresent in a double-stranded conformation includes at least 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 200, 500, 1000, 2000 or5000 nucleotides or includes all of the nucleotides in a cDNA or othertarget nucleic acid sequence being represented in the dsRNA.

In some embodiments, the dsRNA does not contain any single strandedregions, such as single stranded ends, or the dsRNA is a hairpin. Inother embodiments, the dsRNA has one or more single stranded regions oroverhangs. In certain embodiments, RNA/DNA hybrids include a DNA strandor region that is an antisense strand or region (e.g, has at least 70,80, 90, 95, 98, or 100% complementarity to a target nucleic acid) and anRNA strand or region that is a sense strand or region (e.g, has at least70, 80, 90, 95, 98, or 100% identity to a target nucleic acid), and viceversa.

In various embodiments, the RNA/DNA hybrid is made in vitro usingenzymatic or chemical synthetic methods such as those described hereinor those described in WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No.60/130,377, filed Apr. 21, 1999. In other embodiments, a DNA strandsynthesized in vitro is complexed with an RNA strand made in vivo or invitro before, after, or concurrent with the transformation of the DNAstrand into the cell. In yet other embodiments, the dsRNA is a singlecircular nucleic acid containing a sense and an antisense region, or thedsRNA includes a circular nucleic acid and either a second circularnucleic acid or a linear nucleic acid (see, for example, WO 00/63364,filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999.)Exemplary circular nucleic acids include lariat structures in which thefree 5′ phosphoryl group of a nucleotide becomes linked to the 2′hydroxyl group of another nucleotide in a loop back fashion.

In other embodiments, the dsRNA includes one or more modifiednucleotides in which the 2′ position in the sugar contains a halogen(such as fluorine group) or contains an alkoxy group (such as a methoxygroup) which increases the half-life of the dsRNA in vitro or in vivocompared to the corresponding dsRNA in which the corresponding 2′position contains a hydrogen or an hydroxyl group. In yet otherembodiments, the dsRNA includes one or more linkages between adjacentnucleotides other than a naturally-occurring phosphodiester linkage.Examples of such linkages include phosphoramide, phosphorothioate, andphosphorodithioate linkages. The dsRNAs may also be chemically modifiednucleic acid molecules as taught in U.S. Pat. No. 6,673,661. In otherembodiments, the dsRNA contains one or two capped strands, as disclosed,for example, by WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No.60/130,377, filed Apr. 21, 1999.

In other embodiments, the dsRNA can be any of the at least partiallydsRNA molecules disclosed in WO 00/63364, as well as any of the dsRNAmolecules described in U.S. Provisional Application 60/399,998; and U.S.Provisional Application 60/419,532, and PCT/US2003/033466, the teachingof which is hereby incorporated by reference. Any of the dsRNAs may beexpressed in vitro or in vivo using the methods described herein orstandard methods, such as those described in WO 00/63364.

Occupancy

In certain embodiments, antisense compounds are not expected to resultin cleavage or the target nucleic acid via RNase H or to result incleavage or sequestration through the RISC pathway. In certain suchembodiments, antisense activity may result from occupancy, wherein thepresence of the hybridized antisense compound disrupts the activity ofthe target nucleic acid. In certain such embodiments, the antisensecompound may be uniformly modified or may comprise a mix ofmodifications and/or modified and unmodified nucleosides.

Target Nucleic Acids, Target Regions and Nucleotide Sequences

Nucleotide sequences that encode ANGPTL3 include, without limitation,the following: the human sequence as set forth in GenBank Accession No.NM_014495.2 (incorporated herein as SEQ ID NO: 1) or GenBank AccessionNo. NT_032977.9 nucleotides 33032001 to 33046000 (incorporated herein asSEQ ID NO: 2). It is understood that the sequence set forth in each SEQID NO in the Examples contained herein is independent of anymodification to a sugar moiety, an internucleoside linkage, or anucleobase. As such, antisense compounds defined by a SEQ ID NO cancomprise, independently, one or more modifications to a sugar moiety, aninternucleoside linkage, or a nucleobase. Antisense compounds describedby Isis Number (Isis No) indicate a combination ofnucleobase sequenceand motif.

In certain embodiments, a target region is a structurally defined regionof the target nucleic acid. For example, a target region can encompass a3′ UTR, a 5′ UTR, an exon, an intron, an exon/intron junction, a codingregion, a translation initiation region, translation termination region,or other defined nucleic acid region. The structurally defined regionsfor ANGPTL3 can be obtained by accession number from sequence databasessuch as NCBI and such information is incorporated herein by reference.In certain embodiments, a target region can encompass the sequence froma 5′ target site of one target segment within the target region to a 3′target site of another target segment within the target region.

In certain embodiments, a “target segment” is a smaller, sub-portion ofa target region within a nucleic acid. For example, a target segment canbe the sequence of nucleotides of a target nucleic acid to which one ormore antisense compound is targeted. “5′ target site” or “5′ start stie”refers to the 5′-most nucleotide of a target segment. “3′ target site”or “3′ stop site” refers to the 3′-most nucleotide of a target segment.

Targeting includes determination of at least one target segment to whichan antisense compound hybridizes, such that a desired effect occurs. Incertain embodiments, the desired effect is a reduction in mRNA targetnucleic acid levels. In certain embodiments, the desired effect isreduction of levels of protein encoded by the target nucleic acid or aphenotypic change associated with the target nucleic acid.

A target region can contain one or more target segments. Multiple targetsegments within a target region can be overlapping. Alternatively, theycan be non-overlapping. In certain embodiments, target segments within atarget region are separated by no more than about 300 nucleotides. Incertain embodiments, target segments within a target region areseparated by a number of nucleotides that is, is about, is no more than,is no more than about, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30,20, or 10 nucleotides on the target nucleic acid, or is a range definedby any two of the preceding values. In certain embodiments, targetsegments within a target region are separated by no more than, or nomore than about, 5 nucleotides on the target nucleic acid. In certainembodiments, target segments are contiguous. Contemplated are targetregions defined by a range having a starting nucleic acid that is any ofthe 5′ target sites or 3′ target sites listed herein.

Suitable target segments can be found within a 5′ UTR, a coding region,a 3′ UTR, an intron, an exon, or an exon/intron junction. Targetsegments containing a start codon or a stop codon are also suitabletarget segments. A suitable target segment can specifically exclude acertain structurally defined region such as the start codon or stopcodon.

The determination of suitable target segments can include a comparisonof the sequence of a target nucleic acid to other sequences throughoutthe genome. For example, the BLAST algorithm can be used to identifyregions of similarity amongst different nucleic acids. This comparisoncan prevent the selection of antisense compound sequences that canhybridize in a non-specific manner to sequences other than a selectedtarget nucleic acid (i.e., non-target or off-target sequences).

There can be variation in activity (e.g., as defined by percentreduction of target nucleic acid levels) of the antisense compoundswithin an active target region. In certain embodiments, reductions inANGPTL3 mRNA levels are indicative of inhibition of ANGPTL3 proteinexpression. Reductions in levels of an ANGPTL3 protein are alsoindicative of inhibition of target mRNA expression. Further, phenotypicchanges, such as a reduction of the level of cholesterol, LDL,triglyceride, or glucose, can be indicative of inhibition of ANGPTL3mRNA and/or protein expression.

Hybridization

In some embodiments, hybridization occurs between an antisense compounddisclosed herein and an ANGPTL3 nucleic acid. The most common mechanismof hybridization involves hydrogen bonding (e.g., Watson-Crick,Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementarynucleobases of the nucleic acid molecules.

Hybridization can occur under varying conditions. Stringent conditionsare sequence-dependent and are determined by the nature and compositionof the nucleic acid molecules to be hybridized.

Methods of determining whether a sequence is specifically hybridizableto a target nucleic acid are well known in the art (Sambrook andRussell, Molecular Cloning: A Laboratory Manual, 3^(rd) Ed., 2001). Incertain embodiments, the antisense compounds provided herein arespecifically hybridizable with an ANGPTL3 nucleic acid.

Complementarity

An antisense compound and a target nucleic acid are complementary toeach other when a sufficient number of nucleobases of the antisensecompound can hydrogen bond with the corresponding nucleobases of thetarget nucleic acid, such that a desired effect will occur (e.g.,antisense inhibition of a target nucleic acid, such as an ANGPTL3nucleic acid).

An antisense compound can hybridize over one or more segments of anANGPTL3 nucleic acid such that intervening or adjacent segments are notinvolved in the hybridization event (e.g., a loop structure, mismatch orhairpin structure).

In certain embodiments, the antisense compounds provided herein, or aspecified portion thereof, are, or are at least, 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% complementary to an ANGPTL3 nucleic acid, a target region, targetsegment, or specified portion thereof. In certain embodiments, theantisense compounds provided herein, or a specified portion thereof,are, or are at least, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to thesequence of one or more of SEQ ID NOs: 1-2. Percent complementarity ofan antisense compound with a target nucleic acid can be determined usingroutine methods.

For example, an antisense compound in which 18 of 20 nucleobases of theantisense compound are complementary to a target region, and wouldtherefore specifically hybridize, would represent 90 percentcomplementarity. In this example, the remaining noncomplementarynucleobases can be clustered or interspersed with complementarynucleobases and need not be contiguous to each other or to complementarynucleobases. As such, an antisense compound which is 18 nucleobases inlength having 4 (four) noncomplementary nucleobases which are flanked bytwo regions of complete complementarity with the target nucleic acidwould have 77.8% overall complementarity with the target nucleic acidand would thus fall within the scope of the present invention. Percentcomplementarity of an antisense compound with a region of a targetnucleic acid can be determined routinely using BLAST programs (basiclocal alignment search tools) and PowerBLAST programs known in the art(Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden,Genome Res., 1997, 7, 649 656). Percent homology, sequence identity orcomplementarity, can be determined by, for example, the Gap program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, Madison Wis.), using defaultsettings, which uses the algorithm of Smith and Waterman (Adv. Appl.Math., 1981, 2, 482 489).

In certain embodiments, the antisense compounds provided herein, orspecified portions thereof, are fully complementary (i.e. 100%complementary) to a target nucleic acid, or specified portion thereof.For example, an antisense compound can be fully complementary to anANGPTL3 nucleic acid, or a target region, or a target segment or targetsequence thereof. As used herein, “fully complementary” means eachnucleobase of an antisense compound is capable of precise base pairingwith the corresponding nucleobases of a target nucleic acid. Forexample, a 20 nucleobase antisense compound is fully complementary to atarget sequence that is 400 nucleobases long, so long as there is acorresponding 20 nucleobase portion of the target nucleic acid that isfully complementary to the antisense compound. Fully complementary canalso be used in reference to a specified portion of the first and/or thesecond nucleic acid. For example, a 20 nucleobase portion of a 30nucleobase antisense compound can be “fully complementary” to a targetsequence that is 400 nucleobases long. The 20 nucleobase portion of the30 nucleobase oligonucleotide is fully complementary to the targetsequence if the target sequence has a corresponding 20 nucleobaseportion wherein each nucleobase is complementary to the 20 nucleobaseportion of the antisense compound. At the same time, the entire 30nucleobase antisense compound can be fully complementary to the targetsequence, depending on whether the remaining 10 nucleobases of theantisense compound are also complementary to the target sequence.

The location of a non-complementary nucleobase can be at the 5′ end or3′ end of the antisense compound. Alternatively, the non-complementarynucleobase or nucleobases can be at an internal position of theantisense compound. When two or more non-complementary nucleobases arepresent, they can be either contiguous (i.e. linked) or non-contiguous.In one embodiment, a non-complementary nucleobase is located in the wingsegment of a gapmer antisense oligonucleotide.

In certain embodiments, antisense compounds that are, or are up to 10,12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprise nomore than 4, no more than 3, no more than 2, or no more than 1non-complementary nucleobase(s) relative to a target nucleic acid, suchas an ANGPTL3 nucleic acid, or specified portion thereof.

In certain embodiments, antisense compounds that are, or are up to 10,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,or 30 nucleobases in length comprise no more than 6, no more than 5, nomore than 4, no more than 3, no more than 2, or no more than 1non-complementary nucleobase(s) relative to a target nucleic acid, suchas an ANGPTL3 nucleic acid, or specified portion thereof.

The antisense compounds provided herein also include those which arecomplementary to a portion of a target nucleic acid. As used herein,“portion” refers to a defined number of contiguous (i.e. linked)nucleobases within a region or segment of a target nucleic acid. A“portion” can also refer to a defined number of contiguous nucleobasesof an antisense compound. In certain embodiments, the antisensecompounds, are complementary to at least an 8 nucleobase portion of atarget segment. In certain embodiments, the antisense compounds arecomplementary to at least a 10 nucleobase portion of a target segment.In certain embodiments, the antisense compounds are complementary to atleast a 15 nucleobase portion of a target segment. Also contemplated areantisense compounds that are complementary to at least an 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of atarget segment, or a range defined by any two of these values.

Identity

The antisense compounds provided herein can also have a defined percentidentity to a particular nucleotide sequence, SEQ ID NO, or the sequenceof a compound represented by a specific Isis number, or portion thereof.As used herein, an antisense compound is identical to the sequencedisclosed herein if it has the same nucleobase pairing ability. Forexample, a RNA which contains uracil in place of thymidine in adisclosed DNA sequence would be considered identical to the DNA sequencesince both uracil and thymidine pair with adenine. Shortened andlengthened versions of the antisense compounds described herein as wellas compounds having non-identical bases relative to the antisensecompounds provided herein also are contemplated. The non-identical basescan be adjacent to each other or dispersed throughout the antisensecompound. Percent identity of an antisense compound is calculatedaccording to the number of bases that have identical base pairingrelative to the sequence to which it is being compared.

In certain embodiments, the antisense compounds, or portions thereof,are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to one or more of the antisense compounds or SEQ ID NOs, or aportion thereof, disclosed herein.

Modifications

A nucleoside is a base-sugar combination. The nucleobase (also known asbase) portion of the nucleoside is normally a heterocyclic base moiety.Nucleotides are nucleosides that further include a phosphate groupcovalently linked to the sugar portion of the nucleoside. For thosenucleosides that include a pentofuranosyl sugar, the phosphate group canbe linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar.Oligonucleotides are formed through the covalent linkage of adjacentnucleosides to one another, to form a linear polymeric oligonucleotide.Within the oligonucleotide structure, the phosphate groups are commonlyreferred to as forming the internucleoside linkages of theoligonucleotide.

Modifications to antisense compounds encompass substitutions or changesto internucleoside linkages, sugar moieties, or nucleobases. Modifiedantisense compounds are often preferred over native forms because ofdesirable properties such as, for example, enhanced cellular uptake,enhanced affinity for nucleic acid target, increased stability in thepresence of nucleases, or increased inhibitory activity.

Chemically modified nucleosides can also be employed to increase thebinding affinity of a shortened or truncated antisense oligonucleotidefor its target nucleic acid. Consequently, comparable results can oftenbe obtained with shorter antisense compounds that have such chemicallymodified nucleosides.

Modified Internucleoside Linkages

The naturally occurring internucleoside linkage of RNA and DNA is a 3′to 5′ phosphodiester linkage. Antisense compounds having one or moremodified, i.e. non-naturally occurring, internucleoside linkages areoften selected over antisense compounds having naturally occurringinternucleoside linkages because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for target nucleicacids, and increased stability in the presence of nucleases.

Oligonucleotides having modified internucleoside linkages includeinternucleoside linkages that retain a phosphorus atom as well asinternucleoside linkages that do not have a phosphorus atom.Representative phosphorus containing internucleoside linkages include,but are not limited to, phosphodiesters, phosphotriesters,methylphosphonates, phosphoramidate, and phosphorothioates. Methods ofpreparation of phosphorous-containing and non-phosphorous-containinglinkages are well known.

In certain embodiments, antisense compounds targeted to an ANGPTL3nucleic acid comprise one or more modified internucleoside linkages. Incertain embodiments, the modified internucleoside linkages arephosphorothioate linkages. In certain embodiments, each internucleosidelinkage of an antisense compound is a phosphorothioate internucleosidelinkage.

In certain embodiments, oligonucleotides comprise modifiedinternucleoside linkages arranged along the oligonucleotide or regionthereof in a defined pattern or modified internucleoside linkage motif.In certain embodiments, internucleoside linkages are arranged in agapped motif. In such embodiments, the internucleoside linkages in eachof two wing regions are different from the internucleoside linkages inthe gap region. In certain embodiments the internucleoside linkages inthe wings are phosphodiester and the internucleoside linkages in the gapare phosphorothioate. The nucleoside motif is independently selected, sosuch oligonucleotides having a gapped internucleoside linkage motif mayor may not have a gapped nucleoside motif and if it does have a gappednucleoside motif, the wing and gap lengths may or may not be the same.

In certain embodiments, oligonucleotides comprise a region having analternating internucleoside linkage motif. In certain embodiments,oligonucleotides of the present invention comprise a region of uniformlymodified internucleoside linkages. In certain such embodiments, theoligonucleotide comprises a region that is uniformly linked byphosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide is uniformly linked by phosphorothioate. In certainembodiments, each internucleoside linkage of the oligonucleotide isselected from phosphodiester and phosphorothioate. In certainembodiments, each internucleoside linkage of the oligonucleotide isselected from phosphodiester and phosphorothioate and at least oneinternucleoside linkage is phosphorothioate.

In certain embodiments, the oligonucleotide comprises at least 6phosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide comprises at least 8 phosphorothioate internucleosidelinkages. In certain embodiments, the oligonucleotide comprises at least10 phosphorothioate internucleoside linkages. In certain embodiments,the oligonucleotide comprises at least one block of at least 6consecutive phosphorothioate internucleoside linkages. In certainembodiments, the oligonucleotide comprises at least one block of atleast 8 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least one block ofat least 10 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least block of atleast one 12 consecutive phosphorothioate internucleoside linkages. Incertain such embodiments, at least one such block is located at the 3′end of the oligonucleotide. In certain such embodiments, at least onesuch block is located within 3 nucleosides of the 3′ end of theoligonucleotide.

In certain embodiments, oligonucleotides comprise one or moremethylphosponate linkages. In certain embodiments, oligonucleotideshaving a gapmer nucleoside motif comprise a linkage motif comprising allphosphorothioate linkages except for one or two methylphosponatelinkages. In certain embodiments, one methylphosponate linkage is in thecentral gap of an oligonucleotide having a gapmer nucleoside motif.

In certain embodiments, it is desirable to arrange the number ofphosphorothioate internucleoside linkages and phosphodiesterinternucleoside linkages to maintain nuclease resistance. In certainembodiments, it is desirable to arrange the number and position ofphosphorothioate internucleoside linkages and the number and position ofphosphodiester internucleoside linkages to maintain nuclease resistance.In certain embodiments, the number of phosphorothioate internucleosidelinkages may be decreased and the number of phosphodiesterinternucleoside linkages may be increased. In certain embodiments, thenumber of phosphorothioate internucleoside linkages may be decreased andthe number of phosphodiester internucleoside linkages may be increasedwhile still maintaining nuclease resistance. In certain embodiments itis desirable to decrease the number of phosphorothioate internucleosidelinkages while retaining nuclease resistance. In certain embodiments itis desirable to increase the number of phosphodiester internucleosidelinkages while retaining nuclease resistance.

Modified Sugar Moieties

Antisense compounds of the invention can optionally contain one or morenucleosides wherein the sugar group has been modified. Such sugarmodified nucleosides may impart enhanced nuclease stability, increasedbinding affinity, or some other beneficial biological property to theantisense compounds. In certain embodiments, nucleosides comprisechemically modified ribofuranose ring moieties. Examples of chemicallymodified ribofuranose rings include without limitation, addition ofsubstitutent groups (including 5′ and 2′ substituent groups, bridging ofnon-geminal ring atoms to form bicyclic nucleic acids (BNA), replacementof the ribosyl ring oxygen atom with S, N(R), or C(R₁)(R₂) (R, R₁ and R₂are each independently H, C₁-C₁₂ alkyl or a protecting group) andcombinations thereof. Examples of chemically modified sugars include2′-F-5′-methyl substituted nucleoside (see PCT International ApplicationWO 2008/101157 Published on Aug. 21, 2008 for other disclosed 5′,2′-bissubstituted nucleosides) or replacement of the ribosyl ring oxygen atomwith S with further substitution at the 2′-position (see published U.S.Patent Application US2005-0130923, published on Jun. 16, 2005) oralternatively 5′-substitution of a BNA (see PCT InternationalApplication WO 2007/134181 Published on Nov. 22, 2007 wherein LNA issubstituted with for example a 5′-methyl or a 5′-vinyl group).

Examples of nucleosides having modified sugar moieties include withoutlimitation nucleosides comprising 5′-vinyl, 5′-methyl (R or S), 4′-S,2′-F, 2′-OCH₃, 2′-OCH₂CH₃, 2′-OCH₂CH₂F and 2′-O(CH₂)₂OCH₃ substituentgroups. The substituent at the 2′ position can also be selected fromallyl, amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, OCF₃, OCH₂F,O(CH₂)₂SCH₃, O(CH₂)₂—O—N(R_(m))(R_(n)), O—CH₂—C(═O)—N(R_(m))(R_(n)), andO—CH₂—C(═O)—N(R₁)—(CH₂)₂—N(R_(m))(R_(n)), where each R_(l), R_(m) andR_(n) is, independently, H or substituted or unsubstituted C₁-C₁₀ alkyl.

As used herein, “bicyclic nucleosides” refer to modified nucleosidescomprising a bicyclic sugar moiety. Examples of bicyclic nucleic acids(BNAs) include without limitation nucleosides comprising a bridgebetween the 4′ and the 2′ ribosyl ring atoms. In certain embodiments,antisense compounds provided herein include one or more BNA nucleosideswherein the bridge comprises one of the formulas: 4′-(CH₂)—O-2′ (LNA);4′-(CH₂)—S-2′; 4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ and4′-CH(CH₂OCH₃)—O-2′ (and analogs thereof see U.S. Pat. No. 7,399,845,issued on Jul. 15, 2008); 4′-C(CH₃)(CH₃)—O-2′ (and analogs thereof seePCT/US2008/068922 published as WO 2009/006478, published Jan. 8, 2009);4′-CH₂—N(OCH₃)-2′ (and analogs thereof see PCT/US2008/064591 publishedas WO/2008/150729, published Dec. 11, 2008); 4′-CH₂—O—N(CH₃)-2′ (seepublished U.S. Patent Application US2004-0171570, published Sep. 2,2004); 4′-CH₂—N(R)—O-2′, wherein R is H, C₁-C₁₂ alkyl, or a protectinggroup (see U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008);4′-CH₂—C(H)(CH₃)-2′ (see Zhou et al., J. Org. Chem., 2009, 74, 118-134);and 4′-CH₂—C(═CH₂)-2′ (and analogs thereof see PCT/US2008/066154published as WO 2008/154401, published on Dec. 8, 2008).

Further bicyclic nucleosides have been reported in published literature(see for example: Srivastava et al., J. Am. Chem. Soc., 2007, 129(26)8362-8379; Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372;Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braaschet al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr. Opinion Mol.Ther., 2001, 3, 239-243; Wahlestedt et al., Proc. Natl. Acad. Sci.U.S.A, 2000, 97, 5633-5638; Singh et al., Chem. Commun., 1998, 4,455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Kumar et al.,Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org.Chem., 1998, 63, 10035-10039; U.S. Pat. Nos. 7,741,457; 7,399,845;7,053,207; 7,034,133; 6,794,499; 6,770,748; 6,670,461; 6,525,191;6,268,490; U.S. Patent Publication Nos.: US2008-0039618; US2007-0287831;US2004-0171570; U.S. Patent Application Ser. Nos. 61/097,787;61/026,995; and International applications: WO 2009/006478; WO2008/154401; WO 2008/150729; WO 2009/100320; WO 2011/017521; WO2009/067647; WO 2010/036698; WO 2007/134181; WO 2005/021570; WO2004/106356; WO 99/14226. Each of the foregoing bicyclic nucleosides canbe prepared having one or more stereochemical sugar configurationsincluding for example α-L-ribofuranose and β-D-ribofuranose (see PCTinternational application PCT/DK98/00393, published on Mar. 25, 1999 asWO 99/14226).

As used herein, “monocylic nucleosides” refer to nucleosides comprisingmodified sugar moieties that are not bicyclic sugar moieties. In certainembodiments, the sugar moiety, or sugar moiety analogue, of a nucleosidemay be modified or substituted at any position.

As used herein, “4′-2′ bicyclic nucleoside” or “4′ to 2′ bicyclicnucleoside” refers to a bicyclic nucleoside comprising a furanose ringcomprising a bridge connecting two carbon atoms of the furanose ringconnects the 2′ carbon atom and the 4′ carbon atom of the sugar ring.

In certain embodiments, bicyclic sugar moieties of BNA nucleosidesinclude, but are not limited to, compounds having at least one bridgebetween the 4′ and the 2′ carbon atoms of the pentofuranosyl sugarmoiety including without limitation, bridges comprising 1 or from 1 to 4linked groups independently selected from —[C(R_(a))(R_(b))]_(n)—,—C(R_(a))═C(R_(b))—, —C(R_(a))═N—, —C(═NR_(a))—, —C(═O)—, —C(═S)—, —O—,—Si(R_(a))₂-, —S(═O)_(x)—, and —N(R_(a))—; wherein: x is 0, 1, or 2; nis 1, 2, 3, or 4; each R_(a) and R_(b) is, independently, H, aprotecting group, hydroxyl, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl,C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substitutedC₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, heterocycleradical, substituted heterocycle radical, heteroaryl, substitutedheteroaryl, C₅-C₇ alicyclic radical, substituted C₅-C₇ alicyclicradical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃, COOJ₁, acyl (C(═O)—H),substituted acyl, CN, sulfonyl (S(═O)₂-J₁), or sulfoxyl (S(═O)-J₁); andeach J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl(C(═O)—H), substituted acyl, a heterocycle radical, a substitutedheterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl ora protecting group.

In certain embodiments, the bridge of a bicyclic sugar moiety is,—[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—,—C(R_(a)R_(b))—N(R)—O— or —C(R_(a)R_(b))—O—N(R)—. In certainembodiments, the bridge is 4′-CH₂-2′, 4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′,4′-CH₂—O-2′, 4′-(CH₂)₂—O-2′, 4′-CH₂—O—N(R)-2′ and 4′-CH₂—N(R)—O-2′-wherein each Ris, independently, H, a protecting group or C₁-C₁₂ alkyl.

In certain embodiments, bicyclic nucleosides are further defined byisomeric configuration. For example, a nucleoside comprising a4′-(CH₂)—O-2′ bridge, may be in the α-L configuration or in the β-Dconfiguration. Previously, α-L-methyleneoxy (4′-CH₂—O-2′) BNA's havebeen incorporated into antisense oligonucleotides that showed antisenseactivity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

In certain embodiments, bicyclic nucleosides include those having a 4′to 2′ bridge wherein such bridges include without limitation,α-L-4′-(CH₂)—O-2′, 3-D-4′-CH₂—O-2′, 4′-(CH₂)₂—O-2′, 4′-CH₂—O—N(R)-2′,4′-CH₂—N(R)—O-2′, 4′-CH(CH₃)—O-2′, 4′-CH₂—S-2′, 4′-CH₂—N(R)-2′,4′-CH₂—CH(CH₃)-2′, and 4′-(CH₂)₃-2′, wherein R is H, a protecting groupor C₁-C₁₂ alkyl.

In certain embodiment, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

-Q_(a)-Q_(b)-Q_(c)- is —CH₂—N(R_(c))—CH₂—, —C(═O)—N(R_(c))—CH₂—,—CH₂—O—N(R_(c))—, —CH₂—N(R_(c))—O— or —N(R_(c))—O—CH₂;

R_(c) is C₁-C₁₂ alkyl or an amino protecting group; and

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium.

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

Z_(a) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl, acyl,substituted acyl, substituted amide, thiol or substituted thiol.

In one embodiment, each of the substituted groups, is, independently,mono or poly substituted with substituent groups independently selectedfrom halogen, oxo, hydroxyl, OJ_(c), NJ_(c)J_(d), SJ_(c), N₃,OC(═X)J_(c), and NJ_(e)C(═X)NJ_(c)J_(d), wherein each J_(c), J_(d) andJ_(e) is, independently, H, C₁-C₆ alkyl, or substituted C₁-C₆ alkyl andX is O or NJ_(c).

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

Z_(b) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl orsubstituted acyl (C(═O)—).

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

R_(d) is C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

each q_(a), q_(b), q_(c) and q_(d) is, independently, H, halogen, C₁-C₆alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl, C₁-C₆ alkoxyl,substituted C₁-C₆ alkoxyl, acyl, substituted acyl, C₁-C₆ aminoalkyl orsubstituted C₁-C₆ aminoalkyl;

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium; q_(a), q_(b), q_(e) and q_(f)are each, independently, hydrogen, halogen, C₁-C₁₂ alkyl, substitutedC₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂alkynyl, substituted C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxy, substituted C₁-C₁₂alkoxy, OJ_(j), SJ_(j), SOJ_(j), SO₂J_(j), NJ_(j)J_(k), N₃, CN,C(═O)OJ_(j), C(═O)N_(j)J_(k), C(═O)J_(j), O—C(═O)NJ_(j)J_(k),N(H)C(═NH)NJ_(j)J_(k), N(H)C(═O)NJ_(j)J_(k) or N(H)C(═S)NJ_(j)J_(k);

or q_(e) and q_(f) together are ═C(q_(g))(q_(h));

q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂ alkyl orsubstituted C₁-C₁₂ alkyl.

The synthesis and preparation of adenine, cytosine, guanine,5-methyl-cytosine, thymine and uracil bicyclic nucleosides having a4′-CH₂—O-2′ bridge, along with their oligomerization, and nucleic acidrecognition properties have been described (Koshkin et al., Tetrahedron,1998, 54, 3607-3630). The synthesis of bicyclic nucleosides has alsobeen described in WO 98/39352 and WO 99/14226.

Analogs of various bicyclic nucleosides that have 4′ to 2′ bridginggroups such as 4′-CH₂—O-2′ and 4′-CH₂—S-2′, have also been prepared(Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222).

Preparation of oligodeoxyribonucleotide duplexes comprising bicyclicnucleosides for use as substrates for nucleic acid polymerases has alsobeen described (Wengel et al., WO 99/14226). Furthermore, synthesis of2′-amino-BNA, a novel conformationally restricted high-affinityoligonucleotide analog has been described in the art (Singh et al., J.Org. Chem., 1998, 63, 10035-10039). In addition, 2′-amino- and2′-methylamino-BNA's have been prepared and the thermal stability oftheir duplexes with complementary RNA and DNA strands has beenpreviously reported.

In certain embodiments, bicyclic nucleosides have the formula:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

each q_(i), q_(j), q_(k) and q_(l) is, independently, H, halogen, C₁-C₁₂alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxyl,substituted C₁-C₁₂ alkoxyl, OJ_(j), SJ_(j), SOJ_(j), SO₂J_(j),NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k), C(═O)J_(j),O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k), N(H)C(═O)NJ_(j)J_(k) orN(H)C(═S)NJ_(j)J_(k); and

q_(i) and q_(j) or q_(i) and q_(k) together are ═C(q_(g))(q_(h)),wherein q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂alkyl or substituted C₁-C₁₂ alkyl.

One carbocyclic bicyclic nucleoside having a 4′-(CH₂)₃-2′ bridge and thealkenyl analog bridge 4′-CH═CH—CH₂-2′ have been described (Frier et al.,Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J.Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation ofcarbocyclic bicyclic nucleosides along with their oligomerization andbiochemical studies have also been described (Srivastava et al., J. Am.Chem. Soc. 2007, 129(26), 8362-8379).

In certain embodiments, bicyclic nucleosides include, but are notlimited to, (A) α-L-methyleneoxy (4′-CH₂—O-2′) BNA, (B) β-D-methyleneoxy(4′-CH₂—O-2′) BNA, (C) ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, (D) aminooxy(4′-CH₂—O—N(R)-2′) BNA, (E) oxyamino (4′-CH₂—N(R)—O-2′) BNA, (F)methyl(methyleneoxy) (4′-CH(CH₃)—O-2′) BNA (also referred to asconstrained ethyl or cEt), (G) methylene-thio (4′-CH₂—S-2′) BNA, (H)methylene-amino (4′-CH₂—N(R)-2′) BNA, (I) methyl carbocyclic(4′-CH₂—CH(CH₃)-2′) BNA, (J) propylene carbocyclic (4′-(CH₂)₃-2′) BNA,and (K) vinyl BNA as depicted below.

wherein Bx is the base moiety and R is, independently, H, a protectinggroup, C₁-C₆ alkyl or C₁-C₆ alkoxy.

As used herein, the term “modified tetrahydropyran nucleoside” or“modified THP nucleoside” means a nucleoside having a six-memberedtetrahydropyran “sugar” substituted for the pentofuranosyl residue innormal nucleosides and can be referred to as a sugar surrogate. ModifiedTHP nucleosides include, but are not limited to, what is referred to inthe art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA),manitol nucleic acid (MNA) (see Leumann, Bioorg. Med. Chem., 2002, 10,841-854) or fluoro HNA (F-HNA) having a tetrahydropyranyl ring system asillustrated below.

In certain embodiment, sugar surrogates are selected having the formula:

wherein:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the oligomeric compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to an oligomeric compound oroligonucleotide and the other of T₃ and T₄ is H, a hydroxyl protectinggroup, a linked conjugate group or a 5′ or 3′-terminal group;

q_(i), q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl; and one of R₁ and R₂ is hydrogenand the other is selected from halogen, substituted or unsubstitutedalkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN,wherein X is O, S or NJ₁ and each J₁, J₂ and J₃ is, independently, H orC₁-C₆ alkyl.

In certain embodiments, q_(i), q₂, q₃, q₄, q₅, q₆ and q₇ are each H. Incertain embodiments, at least one of q_(i), q₂, q₃, q₄, q₅, q₆ and q₇ isother than H. In certain embodiments, at least one of q_(i), q₂, q₃, q₄,q₅, q₆ and q₇ is methyl. In certain embodiments, THP nucleosides areprovided wherein one of R₁ and R₂ is F. In certain embodiments, R₁ isfluoro and R₂ is H; R₁ is methoxy and R₂ is H, and R₁ is methoxyethoxyand R₂ is H.

In certain embodiments, sugar surrogates comprise rings having more than5 atoms and more than one heteroatom. For example nucleosides comprisingmorpholino sugar moieties and their use in oligomeric compounds has beenreported (see for example: Braasch et al., Biochemistry, 2002, 41,4503-4510; and U.S. Pat. Nos. 5,698,685; 5,166,315; 5,185,444; and5,034,506). As used here, the term “morpholino” means a sugar surrogatehaving the following formula:

In certain embodiments, morpholinos may be modified, for example byadding or altering various substituent groups from the above morpholinostructure. Such sugar surrogates are referred to herein as “modifiedmorpholinos.”

Combinations of modifications are also provided without limitation, suchas 2′-F-5′-methyl substituted nucleosides (see PCT InternationalApplication WO 2008/101157 published on Aug. 21, 2008 for otherdisclosed 5′, 2′-bis substituted nucleosides) and replacement of theribosyl ring oxygen atom with S and further substitution at the2′-position (see published U.S. Patent Application US2005-0130923,published on Jun. 16, 2005) or alternatively 5′-substitution of abicyclic nucleic acid (see PCT International Application WO 2007/134181,published on Nov. 22, 2007 wherein a 4′-CH₂—O-2′ bicyclic nucleoside isfurther substituted at the 5′ position with a 5′-methyl or a 5′-vinylgroup). The synthesis and preparation of carbocyclic bicyclicnucleosides along with their oligomerization and biochemical studieshave also been described (see, e.g., Srivastava et al., J. Am. Chem.Soc. 2007, 129(26), 8362-8379).

In certain embodiments, antisense compounds comprise one or moremodified cyclohexenyl nucleosides, which is a nucleoside having asix-membered cyclohexenyl in place of the pentofuranosyl residue innaturally occurring nucleosides. Modified cyclohexenyl nucleosidesinclude, but are not limited to those described in the art (see forexample commonly owned, published PCT Application WO 2010/036696,published on Apr. 10, 2010, Robeyns et al., J. Am. Chem. Soc., 2008,130(6), 1979-1984; Horvath et al., Tetrahedron Letters, 2007, 48,3621-3623; Nauwelaerts et al., J. Am. Chem. Soc., 2007, 129(30),9340-9348; Gu et al., Nucleosides, Nucleotides & Nucleic Acids, 2005,24(5-7), 993-998; Nauwelaerts et al., Nucleic Acids Research, 2005,33(8), 2452-2463; Robeyns et al., Acta Crystallographica, Section F:Structural Biology and Crystallization Communications, 2005, F61(6),585-586; Gu et al., Tetrahedron, 2004, 60(9), 2111-2123; Gu et al.,Oligonucleotides, 2003, 13(6), 479-489; Wang et al., J. Org. Chem.,2003, 68, 4499-4505; Verbeure et al., Nucleic Acids Research, 2001,29(24), 4941-4947; Wang et al., J. Org. Chem., 2001, 66, 8478-82; Wanget al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20(4-7),785-788; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; Published PCTapplication, WO 06/047842; and Published PCT Application WO 01/049687;the text of each is incorporated by reference herein, in theirentirety). Certain modified cyclohexenyl nucleosides have Formula X.

wherein independently for each of said at least one cyclohexenylnucleoside analog of Formula X:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the cyclohexenyl nucleoside analog to an antisense compound orone of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to an antisense compound and the otherof T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup, or a 5′- or 3′-terminal group; and

q₁, q₂, q₃, q₄, q₅, q₆, q₇, q₈ and q₉ are each, independently, H, C₁-C₆alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or other sugarsubstituent group.

Many other monocyclic, bicyclic and tricyclic ring systems are known inthe art and are suitable as sugar surrogates that can be used to modifynucleosides for incorporation into oligomeric compounds as providedherein (see for example review article: Leumann, Christian J. Bioorg. &Med. Chem., 2002, 10, 841-854). Such ring systems can undergo variousadditional substitutions to further enhance their activity.

As used herein, “2′-modified sugar” means a furanosyl sugar modified atthe 2′ position. In certain embodiments, such modifications includesubstituents selected from: a halide, including, but not limited tosubstituted and unsubstituted alkoxy, substituted and unsubstitutedthioalkyl, substituted and unsubstituted amino alkyl, substituted andunsubstituted alkyl, substituted and unsubstituted allyl, andsubstituted and unsubstituted alkynyl. In certain embodiments, 2′modifications are selected from substituents including, but not limitedto: O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)F,O(CH₂)_(n)ONH₂, OCH₂C(═O)N(H)CH₃, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, wheren and m are from 1 to about 10. Other 2′-substituent groups can also beselected from: C₁-C₁₂ alkyl, substituted alkyl, alkenyl, alkynyl,alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, F,CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving pharmacokinetic properties, or a group for improving thepharmacodynamic properties of an antisense compound, and othersubstituents having similar properties. In certain embodiments, modifiednucleosides comprise a 2′-MOE side chain (Baker et al., J. Biol. Chem.,1997, 272, 11944-12000). Such 2′-MOE substitution have been described ashaving improved binding affinity compared to unmodified nucleosides andto other modified nucleosides, such as 2′-O-methyl, O-propyl, andO-aminopropyl. Oligonucleotides having the 2′-MOE substituent also havebeen shown to be antisense inhibitors of gene expression with promisingfeatures for in vivo use (Martin, Helv. Chim. Acta, 1995, 78, 486-504;Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc.Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides,1997, 16, 917-926).

As used herein, “2′-modified” or “2′-substituted” refers to a nucleosidecomprising a sugar comprising a substituent at the 2′ position otherthan H or OH. 2′-modified nucleosides, include, but are not limited to,bicyclic nucleosides wherein the bridge connecting two carbon atoms ofthe sugar ring connects the 2′ carbon and another carbon of the sugarring; and nucleosides with non-bridging 2′ substituents, such as allyl,amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, —OCF₃, O—(CH₂)₂—O—CH₃,2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)), orO—CH₂—C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is,independently, H or substituted or unsubstituted C₁-C₁₀ alkyl.2′-modified nucleosides may further comprise other modifications, forexample at other positions of the sugar and/or at the nucleobase.

As used herein, “2′-F” refers to a nucleoside comprising a sugarcomprising a fluoro group at the 2′ position of the sugar ring.

As used herein, “2′-OMe” or “2′-OCH₃”, “2′-O-methyl” or “2′-methoxy”each refers to a nucleoside comprising a sugar comprising an —OCH₃ groupat the 2′ position of the sugar ring.

As used herein, “MOE” or “2′-MOE” or “2′-OCH₂CH₂OCH₃” or“2′-O-methoxyethyl” each refers to a nucleoside comprising a sugarcomprising a —OCH₂CH₂OCH₃ group at the 2′ position of the sugar ring.

Methods for the preparations of modified sugars are well known to thoseskilled in the art. Some representative U.S. patents that teach thepreparation of such modified sugars include without limitation, U.S.:4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137;5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722;5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,670,633;5,700,920; 5,792,847 and 6,600,032 and International ApplicationPCT/US2005/019219, filed Jun. 2, 2005 and published as WO 2005/121371 onDec. 22, 2005, and each of which is herein incorporated by reference inits entirety.

As used herein, “oligonucleotide” refers to a compound comprising aplurality of linked nucleosides. In certain embodiments, one or more ofthe plurality of nucleosides is modified. In certain embodiments, anoligonucleotide comprises one or more ribonucleosides (RNA) and/ordeoxyribonucleosides (DNA).

In nucleotides having modified sugar moieties, the nucleobase moieties(natural, modified or a combination thereof) are maintained forhybridization with an appropriate nucleic acid target.

In certain embodiments, antisense compounds comprise one or morenucleosides having modified sugar moieties. In certain embodiments, themodified sugar moiety is 2′-MOE. In certain embodiments, the 2′-MOEmodified nucleosides are arranged in a gapmer motif. In certainembodiments, the modified sugar moiety is a bicyclic nucleoside having a(4′-CH(CH₃)—O-2′) bridging group. In certain embodiments, the(4′-CH(CH₃)—O-2′) modified nucleosides are arranged throughout the wingsof a gapmer motif.

Modified Nucleobases

Nucleobase (or base) modifications or substitutions are structurallydistinguishable from, yet functionally interchangeable with, naturallyoccurring or synthetic unmodified nucleobases. Both natural and modifiednucleobases are capable of participating in hydrogen bonding. Suchnucleobase modifications can impart nuclease stability, binding affinityor some other beneficial biological property to antisense compounds.Modified nucleobases include synthetic and natural nucleobases such as,for example, 5-methylcytosine (5-me-C). Certain nucleobasesubstitutions, including 5-methylcytosine substitutions, areparticularly useful for increasing the binding affinity of an antisensecompound for a target nucleic acid. For example, 5-methylcytosinesubstitutions have been shown to increase nucleic acid duplex stabilityby 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds.,Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp.276-278).

Additional modified nucleobases include 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl (—C≡C—CH3) uracil and cytosine andother alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosineand thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine.

Heterocyclic base moieties can also include those in which the purine orpyrimidine base is replaced with other heterocycles, for example7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.Nucleobases that are particularly useful for increasing the bindingaffinity of antisense compounds include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.

In certain embodiments, antisense compounds targeted to an ANGPTL3nucleic acid comprise one or more modified nucleobases. In certainembodiments, shortened or gap-widened antisense oligonucleotidestargeted to an ANGPTL3 nucleic acid comprise one or more modifiednucleobases. In certain embodiments, the modified nucleobase is5-methylcytosine. In certain embodiments, each cytosine is a5-methylcytosine.

Compositions and Methods for Formulating Pharmaceutical Compositions

Antisense oligonucleotides can be admixed with pharmaceuticallyacceptable active or inert substance for the preparation ofpharmaceutical compositions or formulations. Compositions and methodsfor the formulation of pharmaceutical compositions are dependent upon anumber of criteria, including, but not limited to, route ofadministration, extent of disease, or dose to be administered. Antisensecompound targeted to an ANGPTL3 nucleic acid can be utilized inpharmaceutical compositions by combining the antisense compound with asuitable pharmaceutically acceptable diluent or carrier. Apharmaceutically acceptable diluent includes phosphate-buffered saline(PBS). PBS is a diluent suitable for use in compositions to be deliveredparenterally. Accordingly, in one embodiment, employed in the methodsdescribed herein is a pharmaceutical composition comprising an antisensecompound targeted to an ANGPTL3 nucleic acid and a pharmaceuticallyacceptable diluent. In certain embodiments, the pharmaceuticallyacceptable diluent is PBS. In certain embodiments, the antisensecompound is an antisense oligonucleotide.

Pharmaceutical compositions comprising antisense compounds encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other oligonucleotide which, upon administration to an animal,including a human, is capable of providing (directly or indirectly) thebiologically active metabolite or residue thereof. Accordingly, forexample, the disclosure is also drawn to pharmaceutically acceptablesalts of antisense compounds, prodrugs, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents. Suitablepharmaceutically acceptable salts include, but are not limited to,sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at oneor both ends of an antisense compound which are cleaved by endogenousnucleases within the body, to form the active antisense compound.

Conjugated Antisense Compounds

Antisense compounds can be covalently linked to one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the resulting antisense oligonucleotides. Typical conjugategroups include cholesterol moieties and lipid moieties. Additionalconjugate groups include carbohydrates, phospholipids, biotin,phenazine, folate, phenanthridine, anthraquinone, acridine,fluoresceins, rhodamines, coumarins, and dyes.

Antisense compounds can also be modified to have one or more stabilizinggroups that are generally attached to one or both termini of antisensecompounds to enhance properties such as, for example, nucleasestability. Included in stabilizing groups are cap structures. Theseterminal modifications protect the antisense compound having terminalnucleic acids from exonuclease degradation, and can help in deliveryand/or localization within a cell. The cap can be present at the5′-terminus (5′-cap), or at the 3′-terminus (3′-cap), or can be presenton both termini. Cap structures are well known in the art and include,for example, inverted deoxy abasic caps. Further 3′ and 5′-stabilizinggroups that can be used to cap one or both ends of an antisense compoundto impart nuclease stability include those disclosed in WO 03/004602published on Jan. 16, 2003.

In certain embodiments, the present disclosure provides conjugatedantisense compounds represented by the formula:

A-B—C-DE-F)_(q)

wherein

A is the antisense oligonucleotide;

B is the cleavable moiety

C is the conjugate linker

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, conjugated antisense compounds are providedhaving the structure:

In certain embodiments, conjugated antisense compounds are providedhaving the structure:

In certain embodiments, conjugated antisense compounds are providedhaving the structure:

The present disclosure provides the following non-limiting numberedembodiments:

wherein:

-   -   T₂ is a nucleoside, a nucleotide, a monomeric subunit, or an        oligomeric compound.

In embodiments having more than one of a particular variable (e.g., morethan one “m” or “n”), unless otherwise indicated, each such particularvariable is selected independently. Thus, for a structure having morethan one n, each n is selected independently, so they may or may not bethe same as one another.

i. Certain Cleavable Moieties

In certain embodiments, a cleavable moiety is a cleavable bond. Incertain embodiments, a cleavable moiety comprises a cleavable bond. Incertain embodiments, the conjugate group comprises a cleavable moiety.In certain such embodiments, the cleavable moiety attaches to theantisense oligonucleotide. In certain such embodiments, the cleavablemoiety attaches directly to the cell-targeting moiety. In certain suchembodiments, the cleavable moiety attaches to the conjugate linker. Incertain embodiments, the cleavable moiety comprises a phosphate orphosphodiester. In certain embodiments, the cleavable moiety is acleavable nucleoside or nucleoside analog. In certain embodiments, thenucleoside or nucleoside analog comprises an optionally protectedheterocyclic base selected from a purine, substituted purine, pyrimidineor substituted pyrimidine. In certain embodiments, the cleavable moietyis a nucleoside comprising an optionally protected heterocyclic baseselected from uracil, thymine, cytosine, 4-N-benzoylcytosine,5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine,6-N-benzoyladenine, guanine and 2-N-isobutyrylguanine. In certainembodiments, the cleavable moiety is 2′-deoxy nucleoside that isattached to the 3′ position of the antisense oligonucleotide by aphosphodiester linkage and is attached to the linker by a phosphodiesteror phosphorothioate linkage. In certain embodiments, the cleavablemoiety is 2′-deoxy adenosine that is attached to the 3′ position of theantisense oligonucleotide by a phosphodiester linkage and is attached tothe linker by a phosphodiester or phosphorothioate linkage. In certainembodiments, the cleavable moiety is 2′-deoxy adenosine that is attachedto the 3′ position of the antisense oligonucleotide by a phosphodiesterlinkage and is attached to the linker by a phosphodiester linkage.

In certain embodiments, the cleavable moiety is attached to the 3′position of the antisense oligonucleotide. In certain embodiments, thecleavable moiety is attached to the 5′ position of the antisenseoligonucleotide. In certain embodiments, the cleavable moiety isattached to a 2′ position of the antisense oligonucleotide. In certainembodiments, the cleavable moiety is attached to the antisenseoligonucleotide by a phosphodiester linkage. In certain embodiments, thecleavable moiety is attached to the linker by either a phosphodiester ora phosphorothioate linkage. In certain embodiments, the cleavable moietyis attached to the linker by a phosphodiester linkage. In certainembodiments, the conjugate group does not include a cleavable moiety.

In certain embodiments, the cleavable moiety is cleaved after thecomplex has been administered to an animal only after being internalizedby a targeted cell. Inside the cell the cleavable moiety is cleavedthereby releasing the active antisense oligonucleotide. While notwanting to be bound by theory it is believed that the cleavable moietyis cleaved by one or more nucleases within the cell. In certainembodiments, the one or more nucleases cleave the phosphodiester linkagebetween the cleavable moiety and the linker. In certain embodiments, thecleavable moiety has a structure selected from among the following:

wherein each of Bx, Bx₁, Bx₂, and Bx₃ is independently a heterocyclicbase moiety. In certain embodiments, the cleavable moiety has astructure selected from among the following:

i. Certain Linkers

In certain embodiments, the conjugate groups comprise a linker. Incertain such embodiments, the linker is covalently bound to thecleavable moiety. In certain such embodiments, the linker is covalentlybound to the antisense oligonucleotide. In certain embodiments, thelinker is covalently bound to a cell-targeting moiety. In certainembodiments, the linker further comprises a covalent attachment to asolid support. In certain embodiments, the linker further comprises acovalent attachment to a protein binding moiety. In certain embodiments,the linker further comprises a covalent attachment to a solid supportand further comprises a covalent attachment to a protein binding moiety.In certain embodiments, the linker includes multiple positions forattachment of tethered ligands. In certain embodiments, the linkerincludes multiple positions for attachment of tethered ligands and isnot attached to a branching group. In certain embodiments, the linkerfurther comprises one or more cleavable bond. In certain embodiments,the conjugate group does not include a linker.

In certain embodiments, the linker includes at least a linear groupcomprising groups selected from alkyl, amide, disulfide, polyethyleneglycol, ether, thioether (—S—) and hydroxylamino (—O—N(H)—) groups. Incertain embodiments, the linear group comprises groups selected fromalkyl, amide and ether groups. In certain embodiments, the linear groupcomprises groups selected from alkyl and ether groups. In certainembodiments, the linear group comprises at least one phosphorus linkinggroup. In certain embodiments, the linear group comprises at least onephosphodiester group. In certain embodiments, the linear group includesat least one neutral linking group. In certain embodiments, the lineargroup is covalently attached to the cell-targeting moiety and thecleavable moiety. In certain embodiments, the linear group is covalentlyattached to the cell-targeting moiety and the antisense oligonucleotide.In certain embodiments, the linear group is covalently attached to thecell-targeting moiety, the cleavable moiety and a solid support. Incertain embodiments, the linear group is covalently attached to thecell-targeting moiety, the cleavable moiety, a solid support and aprotein binding moiety. In certain embodiments, the linear groupincludes one or more cleavable bond.

In certain embodiments, the linker includes the linear group covalentlyattached to a scaffold group. In certain embodiments, the scaffoldincludes a branched aliphatic group comprising groups selected fromalkyl, amide, disulfide, polyethylene glycol, ether, thioether andhydroxylamino groups. In certain embodiments, the scaffold includes abranched aliphatic group comprising groups selected from alkyl, amideand ether groups. In certain embodiments, the scaffold includes at leastone mono or polycyclic ring system. In certain embodiments, the scaffoldincludes at least two mono or polycyclic ring systems. In certainembodiments, the linear group is covalently attached to the scaffoldgroup and the scaffold group is covalently attached to the cleavablemoiety and the linker. In certain embodiments, the linear group iscovalently attached to the scaffold group and the scaffold group iscovalently attached to the cleavable moiety, the linker and a solidsupport. In certain embodiments, the linear group is covalently attachedto the scaffold group and the scaffold group is covalently attached tothe cleavable moiety, the linker and a protein binding moiety. Incertain embodiments, the linear group is covalently attached to thescaffold group and the scaffold group is covalently attached to thecleavable moiety, the linker, a protein binding moiety and a solidsupport. In certain embodiments, the scaffold group includes one or morecleavable bond.

In certain embodiments, the linker includes a protein binding moiety. Incertain embodiments, the protein binding moiety is a lipid such as forexample including but not limited to cholesterol, cholic acid,adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine), a vitamin (e.g., folate, vitamin A,vitamin E, biotin, pyridoxal), a peptide, a carbohydrate (e.g.,monosaccharide, disaccharide, trisaccharide, tetrasaccharide,oligosaccharide, polysaccharide), an endosomolytic component, a steroid(e.g., uvaol, hecigenin, diosgenin), a terpene (e.g., triterpene, e.g.,sarsasapogenin, friedelin, epifriedelanol derivatized lithocholic acid),or a cationic lipid. In certain embodiments, the protein binding moietyis a C16 to C22 long chain saturated or unsaturated fatty acid,cholesterol, cholic acid, vitamin E, adamantane or 1-pentafluoropropyl.

In certain embodiments, a linker has a structure selected from among:

wherein each n is, independently, from 1 to 20; and p is from 1 to 6.

In certain embodiments, a linker has a structure selected from among:

wherein each n is, independently, from 1 to 20.

In certain embodiments, a linker has a structure selected from among:

wherein n is from 1 to 20.

In certain embodiments, a linker has a structure selected from among:

-   -   wherein each L is, independently, a phosphorus linking group or        a neutral linking group; and    -   each n is, independently, from 1 to 20.

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

wherein n is from 1 to 20.

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, the conjugate linker has the structure:

In certain embodiments, the conjugate linker has the structure:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.

ii. Certain Cell-Targeting Moieties

In certain embodiments, conjugate groups comprise cell-targetingmoieties. Certain such cell-targeting moieties increase cellular uptakeof antisense compounds. In certain embodiments, cell-targeting moietiescomprise a branching group, one or more tether, and one or more ligand.In certain embodiments, cell-targeting moieties comprise a branchinggroup, one or more tether, one or more ligand and one or more cleavablebond.

1. Certain Branching Groups

In certain embodiments, the conjugate groups comprise a targeting moietycomprising a branching group and at least two tethered ligands. Incertain embodiments, the branching group attaches the conjugate linker.In certain embodiments, the branching group attaches the cleavablemoiety. In certain embodiments, the branching group attaches theantisense oligonucleotide. In certain embodiments, the branching groupis covalently attached to the linker and each of the tethered ligands.In certain embodiments, the branching group comprises a branchedaliphatic group comprising groups selected from alkyl, amide, disulfide,polyethylene glycol, ether, thioether and hydroxylamino groups. Incertain embodiments, the branching group comprises groups selected fromalkyl, amide and ether groups. In certain embodiments, the branchinggroup comprises groups selected from alkyl and ether groups. In certainembodiments, the branching group comprises a mono or polycyclic ringsystem. In certain embodiments, the branching group comprises one ormore cleavable bond. In certain embodiments, the conjugate group doesnot include a branching group.

In certain embodiments, a branching group has a structure selected fromamong:

wherein each n is, independently, from 1 to 20;

j is from 1 to 3; and

m is from 2 to 6.

In certain embodiments, a branching group has a structure selected fromamong:

wherein each n is, independently, from 1 to 20; and

m is from 2 to 6.

In certain embodiments, a branching group has a structure selected fromamong:

In certain embodiments, a branching group has a structure selected fromamong:

wherein each A₁ is independently, O, S, C═O or NH; and

each n is, independently, from 1 to 20.

In certain embodiments, a branching group has a structure selected fromamong:

-   -   wherein each A₁ is independently, O, S, C═O or NH; and    -   each n is, independently, from 1 to 20.

In certain embodiments, a branching group has a structure selected fromamong:

wherein A₁ is O, S, C═O or NH; and

each n is, independently, from 1 to 20.

In certain embodiments, a branching group has a structure selected fromamong:

In certain embodiments, a branching group has a structure selected fromamong:

In certain embodiments, a branching group has a structure selected fromamong:

2. Certain Tethers

In certain embodiments, conjugate groups comprise one or more tetherscovalently attached to the branching group. In certain embodiments,conjugate groups comprise one or more tethers covalently attached to thelinking group. In certain embodiments, each tether is a linear aliphaticgroup comprising one or more groups selected from alkyl, ether,thioether, disulfide, amide and polyethylene glycol groups in anycombination. In certain embodiments, each tether is a linear aliphaticgroup comprising one or more groups selected from alkyl, substitutedalkyl, ether, thioether, disulfide, amide, phosphodiester andpolyethylene glycol groups in any combination. In certain embodiments,each tether is a linear aliphatic group comprising one or more groupsselected from alkyl, ether and amide groups in any combination. Incertain embodiments, each tether is a linear aliphatic group comprisingone or more groups selected from alkyl, substituted alkyl,phosphodiester, ether and amide groups in any combination. In certainembodiments, each tether is a linear aliphatic group comprising one ormore groups selected from alkyl and phosphodiester in any combination.In certain embodiments, each tether comprises at least one phosphoruslinking group or neutral linking group.

In certain embodiments, the tether includes one or more cleavable bond.In certain embodiments, the tether is attached to the branching groupthrough either an amide or an ether group. In certain embodiments, thetether is attached to the branching group through a phosphodiestergroup. In certain embodiments, the tether is attached to the branchinggroup through a phosphorus linking group or neutral linking group. Incertain embodiments, the tether is attached to the branching groupthrough an ether group. In certain embodiments, the tether is attachedto the ligand through either an amide or an ether group. In certainembodiments, the tether is attached to the ligand through an ethergroup. In certain embodiments, the tether is attached to the ligandthrough either an amide or an ether group. In certain embodiments, thetether is attached to the ligand through an ether group.

In certain embodiments, each tether comprises from about 8 to about 20atoms in chain length between the ligand and the branching group. Incertain embodiments, each tether group comprises from about 10 to about18 atoms in chain length between the ligand and the branching group. Incertain embodiments, each tether group comprises about 13 atoms in chainlength.

In certain embodiments, a tether has a structure selected from among:

wherein each n is, independently, from 1 to 20; and

each p is from 1 to about 6.

In certain embodiments, a tether has a structure selected from among:

In certain embodiments, a tether has a structure selected from among:

-   -   wherein each n is, independently, from 1 to 20.

In certain embodiments, a tether has a structure selected from among:

wherein L is either a phosphorus linking group or a neutral linkinggroup;

Z₁ is C(═O)O—R₂;

Z₂ is H, C₁-C₆ alkyl or substituted C₁-C₆ alky;

R₂ is H, C₁-C₆ alkyl or substituted C₁-C₆ alky; and

each m₁ is, independently, from 0 to 20 wherein at least one m₁ isgreater than 0 for

each tether.

In certain embodiments, a tether has a structure selected from among:

In certain embodiments, a tether has a structure selected from among:

-   -   wherein Z₂ is H or CH₃; and    -   each m₁ is, independently, from 0 to 20 wherein at least one m₁        is greater than 0 for    -   each tether.

In certain embodiments, a tether has a structure selected from among:

wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.

In certain embodiments, a tether comprises a phosphorus linking group.In certain embodiments, a tether does not comprise any amide bonds. Incertain embodiments, a tether comprises a phosphorus linking group anddoes not comprise any amide bonds.

3. Certain Ligands

In certain embodiments, the present disclosure provides ligands whereineach ligand is covalently attached to a tether. In certain embodiments,each ligand is selected to have an affinity for at least one type ofreceptor on a target cell. In certain embodiments, ligands are selectedthat have an affinity for at least one type of receptor on the surfaceof a mammalian liver cell. In certain embodiments, ligands are selectedthat have an affinity for the hepatic asialoglycoprotein receptor(ASGP-R). In certain embodiments, each ligand is a carbohydrate. Incertain embodiments, each ligand is, independently selected fromgalactose, N-acetyl galactoseamine, mannose, glucose, glucosamone andfucose. In certain embodiments, each ligand is N-acetyl galactoseamine(GalNAc). In certain embodiments, the targeting moiety comprises 2 to 6ligands. In certain embodiments, the targeting moiety comprises 3ligands. In certain embodiments, the targeting moiety comprises 3N-acetyl galactoseamine ligands.

In certain embodiments, the ligand is a carbohydrate, carbohydratederivative, modified carbohydrate, multivalent carbohydrate cluster,polysaccharide, modified polysaccharide, or polysaccharide derivative.In certain embodiments, the ligand is an amino sugar or a thio sugar.For example, amino sugars may be selected from any number of compoundsknown in the art, for example glucosamine, sialic acid,α-D-galactosamine, N-Acetylgalactosamine,2-acetamido-2-deoxy-D-galactopyranose (GalNAc),2-Amino-3-O—[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose (β-muramicacid), 2-Deoxy-2-methylamino-L-glucopyranose,4,6-Dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose,2-Deoxy-2-sulfoamino-D-glucopyranose and N-sulfo-D-glucosamine, andN-Glycoloyl-α-neuraminic acid. For example, thio sugars may be selectedfrom the group consisting of 5-Thio-β-D-glucopyranose, Methyl2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside,4-Thio-β-D-galactopyranose, and ethyl3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-gluco-heptopyranoside.

In certain embodiments, “GalNac” or “Gal-NAc” refers to2-(Acetylamino)-2-deoxy-D-galactopyranose, commonly referred to in theliterature as N-acetyl galactosamine. In certain embodiments, “N-acetylgalactosamine” refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose. Incertain embodiments, “GalNac” or “Gal-NAc” refers to2-(Acetylamino)-2-deoxy-D-galactopyranose. In certain embodiments,“GalNac” or “Gal-NAc” refers to2-(Acetylamino)-2-deoxy-D-galactopyranose, which includes both theβ-form: 2-(Acetylamino)-2-deoxy-β-D-galactopyranose and α-form:2-(Acetylamino)-2-deoxy-D-galactopyranose. In certain embodiments, boththe β-form: 2-(Acetylamino)-2-deoxy-β-D-galactopyranose and α-form:2-(Acetylamino)-2-deoxy-D-galactopyranose may be used interchangeably.Accordingly, in structures in which one form is depicted, thesestructures are intended to include the other form as well. For example,where the structure for an α-form:2-(Acetylamino)-2-deoxy-D-galactopyranose is shown, this structure isintended to include the other form as well. In certain embodiments. Incertain preferred embodiments, the 0-form2-(Acetylamino)-2-deoxy-D-galactopyranose is the preferred embodiment.

In certain embodiments one or more ligand has a structure selected fromamong:

wherein each R₁ is selected from OH and NHCOOH.

In certain embodiments one or more ligand has a structure selected fromamong:

In certain embodiments one or more ligand has a structure selected fromamong:

In certain embodiments one or more ligand has a structure selected fromamong:

iii. Certain Conjugates

In certain embodiments, conjugate groups comprise the structuralfeatures above. In certain such embodiments, conjugate groups comprisethe following structure:

wherein each n is, independently, from 1 to 20.

In certain such embodiments, conjugate groups comprise the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

wherein each n is, independently, from 1 to 20;

Z is H or a linked solid support;

Q is an antisense compound;

X is O or S; and

Bx is a heterocyclic base moiety.

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups comprise the followingstructure:

In certain such embodiments, conjugate groups comprise the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain embodiments, conjugates do not comprise a pyrrolidine.

b. Certain Conjugated Antisense Compounds

In certain embodiments, the conjugates are bound to a nucleoside of theantisense oligonucleotide at the 2′, 3′, of 5′ position of thenucleoside. In certain embodiments, a conjugated antisense compound hasthe following structure:

A-B—C-DE-F)_(q)

wherein

A is the antisense oligonucleotide;

B is the cleavable moiety

C is the conjugate linker

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, a conjugated antisense compound has thefollowing structure:

A-C-DE-F)_(q)

wherein

A is the antisense oligonucleotide;

C is the conjugate linker

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain such embodiments, the conjugate linker comprises at least onecleavable bond.

In certain such embodiments, the branching group comprises at least onecleavable bond.

In certain embodiments each tether comprises at least one cleavablebond.

In certain embodiments, the conjugates are bound to a nucleoside of theantisense oligonucleotide at the 2′, 3′, of 5′ position of thenucleoside.

In certain embodiments, a conjugated antisense compound has thefollowing structure:

A-B—CE-F)_(q)

wherein

A is the antisense oligonucleotide;

B is the cleavable moiety

C is the conjugate linker

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, the conjugates are bound to a nucleoside of theantisense oligonucleotide at the 2′, 3′, of 5′ position of thenucleoside. In certain embodiments, a conjugated antisense compound hasthe following structure:

A-CE-F)_(q)

wherein

A is the antisense oligonucleotide;

C is the conjugate linker

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, a conjugated antisense compound has thefollowing structure:

A-B-DE-F)_(q)

wherein

A is the antisense oligonucleotide;

B is the cleavable moiety

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, a conjugated antisense compound has thefollowing structure:

A-DE-F)_(q)

wherein

A is the antisense oligonucleotide;

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain such embodiments, the conjugate linker comprises at least onecleavable bond.

In certain embodiments each tether comprises at least one cleavablebond.

In certain embodiments, a conjugated antisense compound has a structureselected from among the following:

In certain embodiments, a conjugated antisense compound has a structureselected from among the following:

In certain embodiments, a conjugated antisense compound has a structureselected from among the following:

In certain embodiments, the conjugated antisense compound has thefollowing structure:

Representative United States patents, United States patent applicationpublications, and international patent application publications thatteach the preparation of certain of the above noted conjugates,conjugated antisense compounds, tethers, linkers, branching groups,ligands, cleavable moieties as well as other modifications includewithout limitation, U.S. Pat. No. 5,994,517, U.S. Pat. No. 6,300,319,U.S. Pat. No. 6,660,720, U.S. Pat. No. 6,906,182, U.S. Pat. No.7,262,177, U.S. Pat. No. 7,491,805, U.S. Pat. No. 8,106,022, U.S. Pat.No. 7,723,509, US 2006/0148740, US 2011/0123520, WO 2013/033230 and WO2012/037254, each of which is incorporated by reference herein in itsentirety.

Representative publications that teach the preparation of certain of theabove noted conjugates, conjugated antisense compounds, tethers,linkers, branching groups, ligands, cleavable moieties as well as othermodifications include without limitation, BIESSEN et al., “TheCholesterol Derivative of a Triantennary Galactoside with High Affinityfor the Hepatic Asialoglycoprotein Receptor: a Potent CholesterolLowering Agent” J. Med. Chem. (1995) 38:1846-1852, BIESSEN et al.,“Synthesis of Cluster Galactosides with High Affinity for the HepaticAsialoglycoprotein Receptor” J. Med. Chem. (1995) 38:1538-1546, LEE etal., “New and more efficient multivalent glyco-ligands forasialoglycoprotein receptor of mammalian hepatocytes” Bioorganic &Medicinal Chemistry (2011) 19:2494-2500, RENSEN et al., “Determinationof the Upper Size Limit for Uptake and Processing of Ligands by theAsialoglycoprotein Receptor on Hepatocytes in Vitro and in Vivo” J.Biol. Chem. (2001) 276(40):37577-37584, RENSEN et al., “Design andSynthesis of Novel N-Acetylgalactosamine-Terminated Glycolipids forTargeting of Lipoproteins to the Hepatic Asialoglycoprotein Receptor” J.Med. Chem. (2004) 47:5798-5808, SLIEDREGT et al., “Design and Synthesisof Novel Amphiphilic Dendritic Galactosides for Selective Targeting ofLiposomes to the Hepatic Asialoglycoprotein Receptor” J. Med. Chem.(1999) 42:609-618, and Valentijn et al., “Solid-phase synthesis oflysine-based cluster galactosides with high affinity for theAsialoglycoprotein Receptor” Tetrahedron, 1997, 53(2), 759-770, each ofwhich is incorporated by reference herein in its entirety.

In certain embodiments, conjugated antisense compounds comprise an RNaseH based oligonucleotide (such as a gapmer) or a splice modulatingoligonucleotide (such as a fully modified oligonucleotide) and anyconjugate group comprising at least one, two, or three GalNAc groups. Incertain embodiments a conjugated antisense compound comprises anyconjugate group found in any of the following references: Lee, CarbohydrRes, 1978, 67, 509-514; Connolly et al., J Biol Chem, 1982, 257,939-945; Pavia et al., Int J Pep Protein Res, 1983, 22, 539-548; Lee etal., Biochem, 1984, 23, 4255-4261; Lee et al., Glycoconjugate J, 1987,4, 317-328; Toyokuni et al., Tetrahedron Lett, 1990, 31, 2673-2676;Biessen et al., J Med Chem, 1995, 38, 1538-1546; Valentijn et al.,Tetrahedron, 1997, 53, 759-770; Kim et al., Tetrahedron Lett, 1997, 38,3487-3490; Lee et al., Bioconjug Chem, 1997, 8, 762-765; Kato et al.,Glycobiol, 2001, 11, 821-829; Rensen et al., J Biol Chem, 2001, 276,37577-37584; Lee et al., Methods Enzymol, 2003, 362, 38-43; Westerlindet al., Glycoconj J, 2004, 21, 227-241; Lee et al., Bioorg Med ChemLett, 2006, 16(19), 5132-5135; Maierhofer et al., Bioorg Med Chem, 2007,15, 7661-7676; Khorev et al., Bioorg Med Chem, 2008, 16, 5216-5231; Leeet al., Bioorg Med Chem, 2011, 19, 2494-2500; Kornilova et al., AnalytBiochem, 2012, 425, 43-46; Pujol et al., Angew Chemie Int Ed Engl, 2012,51, 7445-7448; Biessen et al., J Med Chem, 1995, 38, 1846-1852;Sliedregt et al., J Med Chem, 1999, 42, 609-618; Rensen et al., J MedChem, 2004, 47, 5798-5808; Rensen et al., Arterioscler Thromb Vasc Biol,2006, 26, 169-175; van Rossenberg et al., Gene Ther, 2004, 11, 457-464;Sato et al., J Am Chem Soc, 2004, 126, 14013-14022; Lee et al., J OrgChem, 2012, 77, 7564-7571; Biessen et al., FASEB J, 2000, 14, 1784-1792;Rajur et al., Bioconjug Chem, 1997, 8, 935-940; Duff et al., MethodsEnzymol, 2000, 313, 297-321; Maier et al., Bioconjug Chem, 2003, 14,18-29; Jayaprakash et al., Org Lett, 2010, 12, 5410-5413; Manoharan,Antisense Nucleic Acid Drug Dev, 2002, 12, 103-128; Merwin et al.,Bioconjug Chem, 1994, 5, 612-620; Tomiya et al., Bioorg Med Chem, 2013,21, 5275-5281; International applications WO1998/013381; WO2011/038356;WO1997/046098; WO2008/098788; WO2004/101619; WO2012/037254;WO2011/120053; WO2011/100131; WO2011/163121; WO2012/177947;WO2013/033230; WO2013/075035; WO2012/083185; WO2012/083046;WO2009/082607; WO2009/134487; WO2010/144740; WO2010/148013;WO1997/020563; WO2010/088537; WO2002/043771; WO2010/129709;WO2012/068187; WO2009/126933; WO2004/024757; WO2010/054406;WO2012/089352; WO2012/089602; WO2013/166121; WO2013/165816; U.S. Pat.Nos. 4,751,219; 8,552,163; 6,908,903; 7,262,177; 5,994,517; 6,300,319;8,106,022; 7,491,805; 7,491,805; 7,582,744; 8,137,695; 6,383,812;6,525,031; 6,660,720; 7,723,509; 8,541,548; 8,344,125; 8,313,772;8,349,308; 8,450,467; 8,501,930; 8,158,601; 7,262,177; 6,906,182;6,620,916; 8,435,491; 8,404,862; 7,851,615; Published U.S. PatentApplication Publications US2011/0097264; US2011/0097265; US2013/0004427;US2005/0164235; US2006/0148740; US2008/0281044; US2010/0240730;US2003/0119724; US2006/0183886; US2008/0206869; US2011/0269814;US2009/0286973; US2011/0207799; US2012/0136042; US2012/0165393;US2008/0281041; US2009/0203135; US2012/0035115; US2012/0095075;US2012/0101148; US2012/0128760; US2012/0157509; US2012/0230938;US2013/0109817; US2013/0121954; US2013/0178512; US2013/0236968;US2011/0123520; US2003/0077829; US2008/0108801; and US2009/0203132; eachof which is incorporated by reference in its entirety.

Cell Culture and Antisense Compounds Treatment

The effects of antisense compounds on the level, activity or expressionof ANGPTL3 nucleic acids can be tested in vitro in a variety of celltypes. Cell types used for such analyses are available from commercialvendors (e.g. American Type Culture Collection, Manassas, Va.; Zen-Bio,Inc., Research Triangle Park, N.C.; Clonetics Corporation, Walkersville,Md.) and cells are cultured according to the vendor's instructions usingcommercially available reagents (e.g. Invitrogen Life Technologies,Carlsbad, Calif.). Illustrative cell types include, but are not limitedto, HepG2 cells, Hep3B cells, Huh7 (hepatocellular carcinoma) cells,primary hepatocytes, A549 cells, GM04281 fibroblasts and LLC-MK2 cells.

In Vitro Testing of Antisense Oligonucleotides

Described herein are methods for treatment of cells with antisenseoligonucleotides, which can be modified appropriately for treatment withother antisense compounds.

In general, cells are treated with antisense oligonucleotides when thecells reach approximately 60-80% confluence in culture.

One reagent commonly used to introduce antisense oligonucleotides intocultured cells includes the cationic lipid transfection reagentLIPOFECTIN® (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotidesare mixed with LIPOFECTIN® in OPTI-MEM® 1 (Invitrogen, Carlsbad, Calif.)to achieve the desired final concentration of antisense oligonucleotideand a LIPOFECTIN® concentration that typically ranges 2 to 12 ug/mL per100 nM antisense oligonucleotide.

Another reagent used to introduce antisense oligonucleotides intocultured cells includes LIPOFECTAMINE 2000® (Invitrogen, Carlsbad,Calif.). Antisense oligonucleotide is mixed with LIPOFECTAMINE 2000® inOPTI-MEM® 1 reduced serum medium (Invitrogen, Carlsbad, Calif.) toachieve the desired concentration of antisense oligonucleotide and aLIPOFECTAMINE® concentration that typically ranges 2 to 12 ug/mL per 100nM antisense oligonucleotide.

Another reagent used to introduce antisense oligonucleotides intocultured cells includes Cytofectin® (Invitrogen, Carlsbad, Calif.).Antisense oligonucleotide is mixed with Cytofectin® in OPTI-MEM® 1reduced serum medium (Invitrogen, Carlsbad, Calif.) to achieve thedesired concentration of antisense oligonucleotide and a Cytofectin®concentration that typically ranges 2 to 12 ug/mL per 100 nM antisenseoligonucleotide.

Another reagent used to introduce antisense oligonucleotides intocultured cells includes Oligofectamine™ (Invitrogen Life Technologies,Carlsbad, Calif.). Antisense oligonucleotide is mixed withOligofectamine™ in Opti-MEM™-1 reduced serum medium (Invitrogen LifeTechnologies, Carlsbad, Calif.) to achieve the desired concentration ofoligonucleotide with an Oligofectamine™ to oligonucleotide ratio ofapproximately 0.2 to 0.8 μL per 100 nM.

Another reagent used to introduce antisense oligonucleotides intocultured cells includes FuGENE 6 (Roche Diagnostics Corp., Indianapolis,Ind.). Antisense oligomeric compound was mixed with FuGENE 6 in 1 mL ofserum-free RPMI to achieve the desired concentration of oligonucleotidewith a FuGENE 6 to oligomeric compound ratio of 1 to 4 μL of FuGENE 6per 100 nM.

Another technique used to introduce antisense oligonucleotides intocultured cells includes electroporation (Sambrook and Russell, MolecularCloning: A Laboratory Manual, 3^(rd) Ed., 2001).

Cells are treated with antisense oligonucleotides by routine methods.Cells are typically harvested 16-24 hours after antisenseoligonucleotide treatment, at which time RNA or protein levels of targetnucleic acids are measured by methods known in the art and describedherein. In general, when treatments are performed in multiplereplicates, the data are presented as the average of the replicatetreatments.

The concentration of antisense oligonucleotide used varies from cellline to cell line. Methods to determine the optimal antisenseoligonucleotide concentration for a particular cell line are well knownin the art. Antisense oligonucleotides are typically used atconcentrations ranging from 1 nM to 300 nM when transfected withLIPOFECTAMINE2000®, Lipofectin or Cytofectin. Antisense oligonucleotidesare used at higher concentrations ranging from 625 to 20,000 nM whentransfected using electroporation.

RNA Isolation

RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA.Methods of RNA isolation are well known in the art (Sambrook andRussell, Molecular Cloning: A Laboratory Manual, 3^(rd) Ed., 2001). RNAis prepared using methods well known in the art, for example, using theTRIZOL® Reagent (Invitrogen, Carlsbad, Calif.) according to themanufacturer's recommended protocols.

Analysis of Inhibition of Target Levels or Expression

Inhibition of levels or expression of an ANGPTL3 nucleic acid can beassayed in a variety of ways known in the art (Sambrook and Russell,Molecular Cloning: A Laboratory Manual, 3^(rd) Ed., 2001). For example,target nucleic acid levels can be quantitated by, e.g., Northern blotanalysis, competitive polymerase chain reaction (PCR), or quantitativereal-time PCR. RNA analysis can be performed on total cellular RNA orpoly(A)+ mRNA. Methods of RNA isolation are well known in the art.Northern blot analysis is also routine in the art. Quantitativereal-time PCR can be conveniently accomplished using the commerciallyavailable ABI PRISM® 7600, 7700, or 7900 Sequence Detection System,available from PE-Applied Biosystems, Foster City, Calif. and usedaccording to manufacturer's instructions.

Quantitative Real-Time PCR Analysis of Target RNA Levels

Quantitation of target RNA levels can be accomplished by quantitativereal-time PCR using the ABI PRISM® 7600, 7700, or 7900 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. Methods of quantitative real-time PCRare well known in the art.

Prior to real-time PCR, the isolated RNA is subjected to a reversetranscriptase (RT) reaction, which produces complementary DNA (cDNA)that is then used as the substrate for the real-time PCR amplification.The RT and real-time PCR reactions are performed sequentially in thesame sample well. RT and real-time PCR reagents are obtained fromInvitrogen (Carlsbad, Calif.). RT and real-time-PCR reactions arecarried out by methods well known to those skilled in the art.

Gene (or RNA) target quantities obtained by real time PCR can benormalized using either the expression level of a gene whose expressionis constant, such as cyclophilin A or GADPH or by quantifying total RNAusing RIBOGREEN® (Life Technologies™, Inc. Carlsbad, Calif.).Cyclophilin A or GADPH expression can be quantified by real time PCR, bybeing run simultaneously with the target, multiplexing, or separately.Total RNA can be quantified using RIBOGREEN® RNA quantification reagent.Methods of RNA quantification by RIBOGREEN® are taught in Jones, L. J.,et al, (Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR® 4000instrument (PE Applied Biosystems) can be used to measure RIBOGREEN®fluorescence.

Methods for designing real-time PCR probes and primers are well known inthe art, and can include the use of software such as PRIMER EXPRESS®Software (Applied Biosystems, Foster City, Calif.). Probes and primersused in real-time PCR were designed to hybridize to ANGPTL3 specificsequences and are disclosed in the Examples below. The target specificPCR probes can have FAM covalently linked to the 5′ end and TAMRA or MGBcovalently linked to the 3′ end, where FAM is the fluorescent dye andTAMRA or MGB is the quencher dye.

Analysis of Protein Levels

Antisense inhibition of ANGPTL3 nucleic acids can be assessed bymeasuring ANGPTL3 protein levels. Protein levels of ANGPTL3 can beevaluated or quantitated in a variety of ways well known in the art,such as immunoprecipitation, Western blot analysis (immunoblotting),enzyme-linked immunosorbent assay (ELISA), quantitative protein assays,protein activity assays (for example, caspase activity assays),immunohistochemistry, immunocytochemistry or fluorescence-activated cellsorting (FACS) (Sambrook and Russell, Molecular Cloning: A LaboratoryManual, 3^(rd) Ed., 2001). Antibodies directed to a target can beidentified and obtained from a variety of sources, such as the MSRScatalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can beprepared via conventional monoclonal or polyclonal antibody generationmethods well known in the art.

In Vivo Testing of Antisense Compounds

Antisense compounds, for example, antisense oligonucleotides, are testedin animals to assess their ability to inhibit expression of ANGPTL3 andproduce phenotypic changes. Testing can be performed in normal animals,or in experimental disease models. For administration to animals,antisense oligonucleotides are formulated in a pharmaceuticallyacceptable diluent, such as phosphate-buffered saline. Administrationincludes parenteral routes of administration. Following a period oftreatment with antisense oligonucleotides, RNA is isolated from tissueand changes in ANGPTL3 nucleic acid expression are measured. Changes inANGPTL3 protein levels are also measured.

Certain Indications

In certain embodiments, provided herein are methods of treating anindividual comprising administering one or more pharmaceuticalcompositions as described herein. In certain embodiments, the individualhas a metabolic disease and/or cardiovascular disease. In certainembodiments, the individual has combined hyperlipidemia (e.g., familialor non-familial), hypercholesterolemia (e.g., familial homozygoushypercholesterolemia (HoFH), familial heterozygous hypercholesterolemia(HeFH)), dyslipidemia, lipodystrophy, hypertriglyceridemia (e.g.,heterozygous LPL deficiency, homozygous LPL deficiency), coronary arterydisease (CAD), familial chylomicronemia syndrome (FCS),hyperlipoproteinemia Type IV), metabolic syndrome, non-alcoholic fattyliver disease (NAFLD), nonalcoholic steatohepatitis (NASH), diabetes(e.g., Type 2 diabetes, Type 2 diabetes with dyslipidemia), insulinresistance (e.g., insulin resistance with dyslipidemia), vascular wallthickening, high blood pressure (e.g., pulmonary arterial hypertension),sclerosis (e.g., atherosclerosis, systemic sclerosis, progressive skinsclerosis and proliferative obliterative vasculopathy such as digitalulcers and pulmonary vascular involvement), or a combination thereof.

In certain embodiments, the compounds targeted to ANGPTL3 describedherein modulate lipid and/or energy metabolism in an animal. In certainembodiments, the compounds targeted to ANGPTL3 described herein modulatephysiological markers or phenotypes of hypercholesterolemia,dyslipidemia, lipodystrophy, hypertriglyceridemia, metabolic syndrome,NAFLD, NASH and/or diabetes. For example, administration of thecompounds to animals can modulate one or more of VLDL, non-esterifiedfatty acids (NEFA), LDL, cholesterol, triglyceride, glucose, insulin orANGPTL3 levels. In certain embodiments, the modulation of thephysiological markers or phenotypes can be associated with inhibition ofANGPTL3 by the compounds.

In certain embodiments, the compounds targeted to ANGPTL3 describedherein reduce and/or prevent one or more of hepatic TG accumulation(i.e. hepatic steatosis), atherosclerosis, vascular wall thinkening(e.g., arterial intima-media thickening), combined hyperlipidemia (e.g.,familial or non-familial), hypercholesterolemia (e.g., familialhomozygous hypercholesterolemia (HoFH), familial heterozygoushypercholesterolemia (HeFH)), dyslipidemia, lipodystrophy,hypertriglyceridemia (e.g., heterozygous LPL deficiency, homozygous LPLdeficiency, familial chylomicronemia syndrome (FCS),hyperlipoproteinemia Type IV), metabolic syndrome, non-alcoholic fattyliver disease (NAFLD), nonalcoholic steatohepatitis (NASH), diabetes(e.g., Type 2 diabetes, Type 2 diabetes with dyslipidemia), insulinresistance (e.g., insulin resistance with dyslipidemia), high bloodpressure and sclerosis, or any combination thereof. In certainembodiments, the compounds targeted to ANGPTL3 described herein improveinsulin sensitivity.

In certain embodiments, administration of an antisense compound targetedto an ANGPTL3 nucleic acid results in reduction of ANGPTL3 expression byabout at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95% or at least 99%, or a range defined byany two of these values.

In certain embodiments, pharmaceutical compositions comprising anantisense compound targeted to ANGPTL3 are used for the preparation of amedicament for treating a patient suffering from, or susceptible to, ametabolic disease or cardiovascular disease. In certain embodiments,pharmaceutical compositions comprising an antisense compound targeted toANGPTL3 are used in the preparation of a medicament for treating apatient suffering from, or susceptible to, one or more of combinedhyperlipidemia (e.g., familial or non-familial), hypercholesterolemia(e.g., familial homozygous hypercholesterolemia (HoFH), familialheterozygous hypercholesterolemia (HeFH)), dyslipidemia, lipodystrophy,hypertriglyceridemia (e.g., familial chylomicronemia syndrome (FCS),hyperlipoproteinemia Type IV), metabolic syndrome, non-alcoholic fattyliver disease (NAFLD), nonalcoholic steatohepatitis (NASH), diabetes(e.g., Type 2 diabetes, Type 2 diabetes with dyslipidemia), insulinresistance (e.g., insulin resistance with dyslipidemia), vascular wallthickening, high blood pressure and sclerosis, or a combination thereof.

Administration

In certain embodiments, the compounds and compositions as describedherein are administered parenterally.

In certain embodiments, parenteral administration is by infusion.Infusion can be chronic or continuous or short or intermittent. Incertain embodiments, infused pharmaceutical agents are delivered with apump.

In certain embodiments, parenteral administration is by injection. Theinjection can be delivered with a syringe or a pump. In certainembodiments, the injection is a bolus injection. In certain embodiments,the injection is administered directly to a tissue or organ. In certainembodiments, the injection is subcutaneous.

Certain Combination Therapies

In certain embodiments, a first agent comprising the modifiedoligonucleotide disclosed herein is co-administered with one or moresecondary agents. In certain embodiments, such second agents aredesigned to treat the same disease, disorder or condition as the firstagent described herein. In certain embodiments, such second agents aredesigned to treat a different disease, disorder, or condition as thefirst agent described herein. In certain embodiments, such second agentsare designed to treat an undesired side effect of one or morepharmaceutical compositions as described herein. In certain embodiments,second agents are co-administered with the first agent to treat anundesired effect of the first agent. In certain embodiments, secondagents are co-administered with the first agent to produce acombinational effect. In certain embodiments, second agents areco-administered with the first agent to produce a synergistic effect.

In certain embodiments, a first agent and one or more second agents areadministered at the same time. In certain embodiments, the first agentand one or more second agents are administered at different times. Incertain embodiments, the first agent and one or more second agents areprepared together in a single pharmaceutical formulation. In certainembodiments, the first agent and one or more second agents are preparedseparately.

In certain embodiments, second agents include, but are not limited to aglucose-lowering agent or a lipid-lowering agent. The glucose loweringagent can include, but is not limited to, a therapeutic lifestylechange, PPAR agonist, a dipeptidyl peptidase (IV) inhibitor, a GLP-1analog, insulin or an insulin analog, an insulin secretagogue, a SGLT2inhibitor, a human amylin analog, a biguanide, an alpha-glucosidaseinhibitor, or a combination thereof. The glucose-lowering agent caninclude, but is not limited to metformin, sulfonylurea, rosiglitazone,meglitinide, thiazolidinedione, alpha-glucosidase inhibitor or acombination thereof. The sulfonylurea can be acetohexamide,chlorpropamide, tolbutamide, tolazamide, glimepiride, a glipizide, aglyburide, or a gliclazide. The meglitinide can be nateglinide orrepaglinide. The thiazolidinedione can be pioglitazone or rosiglitazone.The alpha-glucosidase can be acarbose or miglitol. In certainembodiments the lipid lowering therapy can include, but is not limitedto, a therapeutic lifestyle change, niacin, HMG-CoA reductase inhibitor,cholesterol absorption inhibitor, MTP inhibitor (e.g., a small molecule,polypeptide, antibody or antisense compound targeted to MTP), fibrate,PCSK9 inhibitor (e.g., PCSK9 antibodies, polypeptides, small moleculesnucleic acid compounds targeting PCSK9), CETP inhibitor (e.g., smallmolecules such as torcetrapib and anacetrapib, polypeptides, antibodiesor nucleic acid compounds targeted to CETP), apoC-III inhibitor (e.g., asmall molecule, polypeptide, antibody or nucleic acid compounds targetedto apoC-III), apoB inhibitor (e.g., a small molecule, polypeptide,antibody or nucleic acid compounds targeted to apoB), beneficial oilsrich in omega-3 fatty acids, omega-3 fatty acids or any combinationthereof. The HMG-CoA reductase inhibitor can be atorvastatin,rosuvastatin, fluvastatin, lovastatin, pravastatin, simvastatin and thelike. The cholesterol absorption inhibitor can be ezetimibe. The fibratecan be fenofibrate, bezafibrate, ciprofibrate, clofibrate, gemfibroziland the like. The beneficial oil rich in omega-3 fatty acids can bekrill, fish (e.g., Vascepa®), flaxseed oil and the like. The omega-3fatty acid can be ALA, DHA, EPA and the like.

Certain Compounds

Antisense oligonucleotides targeting human ANGPTL3 were described in anearlier publication (see PCT Patent Publication No. WO 2011/085271published Jul. 14, 2011, incorporated by reference herein, in itsentirety). Several oligonucleotides (233676, 233690, 233710, 233717,233721, 233722, 337459, 337460, 337474, 337477, 337478, 337479, 337481,337484, 337487, 337488, 337490, 337491, 337492, 337497, 337498, 337503,337505, 337506, 337508, 337513, 337514, 337516, 337520, 337521, 337525,337526 and 337528) described therein, including the top ten most potentantisense compounds in vitro, were used as benchmarks throughout selectin vitro screens for antisense compounds described hereinbelow and inU.S. Ser. No. 61/920,652. Of the most potent compounds described in WO2011/085271, ISIS 233722 was found to be highly variable in its abilityto inhibit ANGPTL3. According, although initially included in some invitro studies, 233722 was not selected as a benchmark for furtherstudies. Of the previously identified potent in vitro benchmarkcompounds, five (233710, 233717, 337477, 337478, 337479 and 337487) wereselected for testing in vivo, as described hereinbelow, in huANGPTL3transgenic mice to assess the most potent in reducing human mRNAtranscript and protein expression (Example 126). The antisenseoligonucleotide with the highest initial in vivo potency in reducingANGPTL3 levels (233710) was used as a benchmark for in vivo assessmentof the new antisense compounds described hereinbelow.

In certain embodiments, the antisense compounds described herein benefitfrom one or more improved properties relative to the antisense compoundsdescribed in WO 2011/085271 and in U.S. Ser. No. 61/920,652. Theseimproved properties are demonstrated in the examples herein, and anon-exhaustive summary of the examples is provided below for ease ofreference.

In a first screen described herein, about 3000 newly designed 5-10-5 MOEgapmer antisense compounds targeting human ANGPTL3 were tested in Hep3Bcells for their effect on human ANGPTL3 mRNA in vitro (Example 116). ThemRNA inhibition levels of the new antisense compounds were assessed withsome previously designed antisense compounds (233717, 337484, 337487,337492 and 337516) used as benchmarks in select studies. Of the about3000 newly designed antisense compounds from this first screen, about 85antisense compounds were selected for in vitro dose-dependent inhibitionstudies to determine their half maximal inhibitory concentration (IC₅₀)(Examples 117-118). Of the about 85 new antisense compounds tested fortheir half maximal inhibitory concentration (IC₅₀), about 38 antisensecompounds that demonstrated potent dose-dependent reduction of ANGPTL3were selected for in vivo potency and tolerability (ALT and AST) testingin mice (Examples 126-127) with antisense compound 233710 used as abenchmark.

In a second screen described herein, about 2000 newly designed antisensecompounds targeting human ANGPTL3 with a MOE gapmer motif or a mixedmotif (deoxy, 5-10-5 MOE and cET gapmers) were also tested in Hep3Bcells for their effect on human ANGPTL3 mRNA in vitro (Examples119-121). The inhibition levels of the new antisense compounds wereassessed with some previously designed antisense compounds (233717,337487, 337513, 337514 and 337516) used as benchmarks in select studies.Of the about 2000 newly designed antisense compounds from this secondscreen, about 147 antisense compounds were selected for in vitrodose-dependent inhibition studies to determine their half maximalinhibitory concentration (IC₅₀) (Examples 122-125). Of the about 147 newantisense compounds from tested for their half maximal inhibitoryconcentration (IC₅₀), about 73 antisense compounds that demonstratedpotent dose-dependent reduction of ANGPTL3 were selected for in vivopotency and tolerability (e.g., ALT and AST) testing in mice (Examples126-127) with antisense compound 233710 used as a benchmark.

Of the about 111 antisense compounds from screens one and two that weretested for potency and tolerability in mice, 24 were selected for moreextensive tolerability testing in mice by assessing liver metabolicmarkers, such as alanine transaminase (ALT), aspartate transaminase(AST), albumin and bilirubin, as well as kidney metabolic markers BUNand creatinine and organ weight (Example 127).

In parallel with the in vivo murine studies seventeen antisensecompounds were selected for viscosity testing (Example 128). Generally,antisense compounds that were not optimal for viscosity were not takenforward in further studies.

Based on the results of the mice tolerability study, twenty antisensecompounds were selected for in vivo tolerability testing in rats(Example 129). In the rats, liver metabolic markers, such as ALT, AST,albumin and bilirubin, body and organ weights, as well as kidneymetabolic markers, such as BUN, creatinine and total protein/creatinineratio, were measured to determine the tolerability of a compound invivo.

The twenty antisense compounds tested in the rats were also assessed forcross-reactivity to a rhesus monkey ANGPTL3 gene sequence (Example 130).Although the antisense compounds in this study were tested in cynomolgusmonkeys, the cynomolgus monkey ANGPTL3 sequence was not available forcomparison to the sequences of the full-length compounds, therefore thesequences of the antisense compounds were compared to that of theclosely related rhesus monkey. The sequences of eight antisensecompounds were found to have 0-2 mismatches with the rhesus ANGPTL3 genesequence and were further studied in cynomolgus monkeys (Example 130).The eight antisense compounds (ISIS 563580, ISIS 560400, ISIS 567320,ISIS 567321, ISIS 544199, ISIS 567233, ISIS 561011 and ISIS 559277) weretested for inhibition of ANGPTL3 mRNA and protein expression as well astolerability in the monkeys. In the tolerability studies, body weights,liver metabolic markers (ALT, AST and bilirubin), kidney metabolicmarkers (BUN and creatinine), hematology parameters (blood cell counts,hemoglobin and hematocrit), and pro-inflammatory markers (CRP and C3)were measured. Additionally, the full-length oligonucleotideconcentration present in liver and kidney was measured and the ratio offull-length oligonucleotide in the kidney/liver was calculated.

The sequence of a potent and tolerable antisense compound, ISIS 563580,assessed in cynomolgus monkeys was further modified with a GalNAcconjugate and/or changes in the backbone chemistry as shown in Examples113-115 and 131 and evaluated for increase potency.

Accordingly, provided herein are antisense compounds with any one ormore improved characteristics e.g., improved relative to the antisensecompounds described in WO 2011/085271 and in U.S. Ser. No. 61/920,652.In certain embodiments, provided herein are antisense compoundscomprising a modified oligonucleotide as described herein targeted to,or specifically hybridizable with, a region of nucleotides of any one ofSEQ ID NOs: 1-2.

In certain embodiments, certain antisense compounds as described hereinare efficacious by virtue of their potency in inhibiting ANGPTL3expression. In certain embodiments, the compounds or compositionsinhibit ANGPTL3 by at least 40%, at least 45%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90% or at least 95%.

In certain embodiments, certain antisense compounds as described hereinare efficacious by virtue of an in vitro IC₅₀ of less than 20 μM, lessthan 10 μM, less than 8 μM, less than 5 μM, less than 2 μM, less than 1μM, less than 0.9 μM, less than 0.8 μM, less than 0.7 μM, less than 0.6μM, or less than 0.5 μM when tested in human cells, for example, in theHep3B cell line (as described in Examples 117-118 and 122-125). Incertain embodiments, preferred antisense compounds having an IC₅₀<1.0 μMinclude SEQ ID NOs: 15, 20, 24, 34, 35, 36, 37, 42, 43, 44, 47, 50, 51,57, 58, 60, 77, 79, 82, 87, 88, 90, 91, 93, 94, 100, 101, 104, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 169, 170, 177, 188, 209, 210, 211, 212, 213, 214,215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228,229, 230, 231, and 232. In certain embodiments, preferred antisensecompounds having an IC₅₀<0.9 μM include SEQ ID NOs: 15, 20, 35, 36, 42,43, 44, 50, 57, 60, 77, 79, 87, 88, 90, 91, 93, 94, 101, 104, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 177, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218,219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, and232. In certain embodiments, preferred antisense compounds having anIC₅₀<0.8 μM include SEQ ID NOs: 15, 20, 35, 36, 42, 43, 44, 50, 57, 60,77, 79, 87, 88, 90, 91, 93, 94, 101, 104, 110, 111, 112, 113, 114, 115,116, 117, 118, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 177, 209,210, 211, 212, 213, 214, 215, 217, 218, 219, 220, 221, 222, 223, 224,225, 228, 229, 230, 231, and 232. In certain embodiments, preferredantisense compounds having an IC₅₀<0.7 μM include SEQ ID NOs: 15, 20,36, 42, 43, 57, 60, 114, 117, 127, 131, 177, 209, 210, 211, 212, 213,214, 215, 217, 218, 219, 220, 221, 222, 223, 224, 225, 228, 229, 230,231, and 232. In certain embodiments, preferred antisense compoundshaving an IC₅₀<0.6 μM include SEQ ID NOs: 15, 20, 36, 42, 43, 57, 60,114, 117, 127, 131, 177, 209, 210, 211, 212, 213, 215, 217, 218, 219,220, 221, 222, 224, 225, 228, 229, 230, 231, and 232. In certainembodiments, preferred antisense compounds having an IC₅₀<0.5 μM includeSEQ ID NOs: 43, 114, 117, 127, 131, 177, 209, 210, 211, 212, 215, 217,218, 219, 220, 221, 222, 229, 230, and 232.

In certain embodiments, certain antisense compounds as described hereinare efficacious by virtue of having a viscosity of less than 40 cP, lessthan 35 cP, less than 30 cP, less than 25 cP, less than 20 cP, less than15 cP, or less than 10 cP when measured by an assay (as described inExample 128). Oligonucleotides having a viscosity greater than 40 cPwould have less than optimal viscosity. In certain embodiments,preferred antisense compounds having a viscosity <20 cP include SEQ IDNOs: 16, 18, 20, 34, 35, 36, 38, 49, 77, 90, 93, and 94. In certainembodiments, preferred antisense compounds having a viscosity <15 cPinclude SEQ ID NOs: 16, 18, 20, 34, 35, 38, 49, 90, 93, and 94. Incertain embodiments, preferred antisense compounds having a viscosity<10 cP include SEQ ID NOs: 18, 34, 35, 49, 90, 93, and 94.

In certain embodiments, certain antisense compounds as described hereinare highly tolerable, as demonstrated by the in vivo tolerabilitymeasurements described in the examples. In certain embodiments, thecertain antisense compounds as described herein are highly tolerable, asdemonstrated by having an increase in ALT and/or AST value of no morethan 3 fold, 2 fold or 1.5 fold over saline treated animals.

In certain embodiments, certain antisense compounds as described hereinare efficacious by virtue of having one or more of an inhibition potencyof greater than 50%, an in vitro IC₅₀ of less than 1 μM, a viscosity ofless than 20 cP, and no more than a 3 fold increase in ALT and/or AST.

In certain embodiments, ISIS 563580 (SEQ ID NO: 77) is preferred. Thiscompound was found to be a potent inhibitor in ANGPTL3 transgenic miceand the most tolerable antisense compound. It had an acceptableviscosity of about 16.83 cP and an IC₅₀ value of <0.8 μM in vitro. Inmice it had no more than a 3 fold increase in ALT and/or AST levels oversaline treated animals. Also, in monkeys, it was among the mosttolerable and potent compounds in inhibiting ANGPTL3 and had the bestratio of full-length oligonucleotide concentration.

In certain embodiments, ISIS 544199 (SEQ ID NO: 20) is preferred. Thiscompound was found to be a potent and tolerable antisense compound. Ithad an acceptable viscosity of 1.7 cP and an IC₅₀ value of <0.5 μM invitro. In mice it had no more than a 3 fold increase in ALT and/or ASTlevels over saline treated animals. Also, in monkeys, it was among themost potent compounds in inhibiting ANGPTL3 and had a good ratio offull-length oligonucleotide concentration.

In certain embodiments, ISIS 559277 (SEQ ID NO: 110) is preferred. Thiscompound was found to be a potent and tolerable antisense compound. Ithad an IC₅₀ value of <0.8 μM in vitro. In mice it had no more than a 3fold increase in ALT and/or AST levels over saline treated animals.Also, in monkeys, it was among the most potent compounds in inhibitingANGPTL3 and had a good ratio of full-length oligonucleotideconcentration.

In certain embodiments, a GalNAc conjugated antisense compound, ISIS658501 (SEQ ID NO: 4912), is preferred. This antisense compound wasfound to be more potent than its parent compound ISIS 563580 (SEQ ID NO:77) as shown by the inhibition levels.

In certain embodiments, a GalNAc conjugated antisense compound, ISIS703801 (SEQ ID NO: 77), is preferred. This antisense compound was foundto be several fold more potent than its parent compound ISIS 563580 (SEQID NO: 77). ISIS 703801 had an ID50 value of 1 while ISIS 563580 had anID50 value of 6.

In certain embodiments, a GalNAc conjugated antisense compound, ISIS703802 (SEQ ID NO: 77), is preferred. This antisense compound was foundto be several fold more potent than its parent compound ISIS 563580 (SEQID NO: 77). ISIS 703802 had an ID50 value of 0.3 while ISIS 563580 hadan ID50 value of 6.

EXAMPLES

The following examples illustrate certain embodiments of the presentdisclosure and are not limiting. Moreover, where specific embodimentsare provided, the inventors have contemplated generic application ofthose specific embodiments. For example, disclosure of anoligonucleotide having a particular motif provides reasonable supportfor additional oligonucleotides having the same or similar motif. And,for example, where a particular high-affinity modification appears at aparticular position, other high-affinity modifications at the sameposition are considered suitable, unless otherwise indicated.

Example 1: General Method for the Preparation of Phosphoramidites,Compounds 1, 1a and 2

-   -   Bx is a heterocyclic base;

Compounds 1, 1a and 2 were prepared as per the procedures well known inthe art as described in the specification herein (see Seth et al.,Bioorg. Med. Chem., 2011, 21(4), 1122-1125, J. Org. Chem., 2010, 75(5),1569-1581, Nucleic Acids Symposium Series, 2008, 52(1), 553-554); andalso see published PCT International Applications (WO 2011/115818, WO2010/077578, WO2010/036698, WO2009/143369, WO 2009/006478, and WO2007/090071), and U.S. Pat. No. 7,569,686).

Example 2: Preparation of Compound 7

Compounds 3(2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-β-Dgalactopyranose orgalactosamine pentaacetate) is commercially available. Compound 5 wasprepared according to published procedures (Weber et al., J. Med. Chem.,1991, 34, 2692).

Example 3: Preparation of Compound 11

Compounds 8 and 9 are commercially available.

Example 4: Preparation of Compound 18

Compound 11 was prepared as per the procedures illustrated in Example 3.Compound 14 is commercially available. Compound 17 was prepared usingsimilar procedures reported by Rensen et al., J. Med. Chem., 2004, 47,5798-5808.

Example 5: Preparation of Compound 23

Compounds 19 and 21 are commercially available.

Example 6: Preparation of Compound 24

Compounds 18 and 23 were prepared as per the procedures illustrated inExamples 4 and 5.

Example 7: Preparation of Compound 25

Compound 24 was prepared as per the procedures illustrated in Example 6.

Example 8: Preparation of Compound 26

Compound 24 is prepared as per the procedures illustrated in Example 6.

Example 9: General Preparation of Conjugated ASOs Comprising GalNAc₃-1at the 3′ Terminus, Compound 29

-   -   Wherein the protected GalNAc₃-1 has the structure:

The GalNAc₃ cluster portion of the conjugate group GalNAc₃-1(GalNAc₃-1_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. Wherein GalNAc₃-1_(a) has the formula:

The solid support bound protected GalNAc₃-1, Compound 25, was preparedas per the procedures illustrated in Example 7. Oligomeric Compound 29comprising GalNAc₃-1 at the 3′ terminus was prepared using standardprocedures in automated DNA/RNA synthesis (see Dupouy et al., Angew.Chem. Int. Ed., 2006, 45, 3623-3627). Phosphoramidite building blocks,Compounds 1 and 1a were prepared as per the procedures illustrated inExample 1. The phosphoramidites illustrated are meant to berepresentative and not intended to be limiting as other phosphoramiditebuilding blocks can be used to prepare oligomeric compounds having apredetermined sequence and composition. The order and quantity ofphosphoramidites added to the solid support can be adjusted to preparegapped oligomeric compounds as described herein. Such gapped oligomericcompounds can have predetermined composition and base sequence asdictated by any given target.

Example 10: General Preparation Conjugated ASOs Comprising GalNAc₃-1 atthe 5′ Terminus, Compound 34

The Unylinker™ 30 is commercially available. Oligomeric Compound 34comprising a GalNAc₃-1 cluster at the 5′ terminus is prepared usingstandard procedures in automated DNA/RNA synthesis (see Dupouy et al.,Angew. Chem. Int. Ed., 2006, 45, 3623-3627). Phosphoramidite buildingblocks, Compounds 1 and 1a were prepared as per the proceduresillustrated in Example 1. The phosphoramidites illustrated are meant tobe representative and not intended to be limiting as otherphosphoramidite building blocks can be used to prepare an oligomericcompound having a predetermined sequence and composition. The order andquantity of phosphoramidites added to the solid support can be adjustedto prepare gapped oligomeric compounds as described herein. Such gappedoligomeric compounds can have predetermined composition and basesequence as dictated by any given target.

Example 11: Preparation of Compound 39

Compounds 4, 13 and 23 were prepared as per the procedures illustratedin Examples 2, 4, and 5. Compound 35 is prepared using similarprocedures published in Rouchaud et al., Eur. J. Org. Chem., 2011, 12,2346-2353.

Example 12: Preparation of Compound 40

Compound 38 is prepared as per the procedures illustrated in Example 11.

Example 13: Preparation of Compound 44

Compounds 23 and 36 are prepared as per the procedures illustrated inExamples 5 and 11. Compound 41 is prepared using similar procedurespublished in WO 2009082607.

Example 14: Preparation of Compound 45

Compound 43 is prepared as per the procedures illustrated in Example 13.

Example 15: Preparation of Compound 47

Compound 46 is commercially available.

Example 16: Preparation of Compound 53

Compounds 48 and 49 are commercially available. Compounds 17 and 47 areprepared as per the procedures illustrated in Examples 4 and 15.

Example 17: Preparation of Compound 54

Compound 53 is prepared as per the procedures illustrated in Example 16.

Example 18: Preparation of Compound 55

Compound 53 is prepared as per the procedures illustrated in Example 16.

Example 19: General Method for the Preparation of Conjugated ASOsComprising GalNAc₃-1 at the 3′ Position Via Solid Phase Techniques(Preparation of ISIS 647535, 647536 and 651900)

Unless otherwise stated, all reagents and solutions used for thesynthesis of oligomeric compounds are purchased from commercial sources.Standard phosphoramidite building blocks and solid support are used forincorporation nucleoside residues which include for example T, A, G, andC residues. A 0.1 μM solution of phosphoramidite in anhydrousacetonitrile was used for β-D-2′-deoxyribonucleoside and 2′-MOE.

The ASO syntheses were performed on ABI 394 synthesizer (1-2 μmol scale)or on GE Healthcare Bioscience ÄKTA oligopilot synthesizer (40-200 μmolscale) by the phosphoramidite coupling method on an GalNAc₃-1 loadedVIMAD solid support (110 μmol/g, Guzaev et al., 2003) packed in thecolumn. For the coupling step, the phosphoramidites were delivered 4fold excess over the loading on the solid support and phosphoramiditecondensation was carried out for 10 min. All other steps followedstandard protocols supplied by the manufacturer. A solution of 6%dichloroacetic acid in toluene was used for removing dimethoxytrityl(DMT) group from 5′-hydroxyl group of the nucleotide.4,5-Dicyanoimidazole (0.7 M) in anhydrous CH₃CN was used as activatorduring coupling step. Phosphorothioate linkages were introduced bysulfurization with 0.1 μM solution of xanthane hydride in 1:1pyridine/CH₃CN for a contact time of 3 minutes. A solution of 20%tert-butylhydroperoxide in CH₃CN containing 6% water was used as anoxidizing agent to provide phosphodiester internucleoside linkages witha contact time of 12 minutes.

After the desired sequence was assembled, the cyanoethyl phosphateprotecting groups were deprotected using a 1:1 (v/v) mixture oftriethylamine and acetonitrile with a contact time of 45 minutes. Thesolid-support bound ASOs were suspended in aqueous ammonia (28-30 wt %)and heated at 55° C. for 6 h.

The unbound ASOs were then filtered and the ammonia was boiled off. Theresidue was purified by high pressure liquid chromatography on a stronganion exchange column (GE Healthcare Bioscience, Source 30Q, 30 μm,2.54×8 cm, A=100 mM ammonium acetate in 30% aqueous CH₃CN, B=1.5 μM NaBrin A, 0-40% of B in 60 min, flow 14 mL min-1, X=260 nm). The residue wasdesalted by HPLC on a reverse phase column to yield the desired ASOs inan isolated yield of 15-30% based on the initial loading on the solidsupport. The ASOs were characterized by ion-pair-HPLC coupled MSanalysis with Agilent 1100 MSD system.

Antisense oligonucleotides not comprising a conjugate were synthesizedusing standard oligonucleotide synthesis procedures well known in theart.

Using these methods, three separate antisense compounds targeting ApoCIII were prepared. As summarized in Table 17, below, each of the threeantisense compounds targeting ApoC III had the same nucleobase sequence;ISIS 304801 is a 5-10-5 MOE gapmer having all phosphorothioate linkages;ISIS 647535 is the same as ISIS 304801, except that it had a GalNAc₃-1conjugated at its 3′ end; and ISIS 647536 is the same as ISIS 647535except that certain internucleoside linkages of that compound arephosphodiester linkages. As further summarized in Table 17, two separateantisense compounds targeting SRB-1 were synthesized. ISIS 440762 was a2-10-2 cEt gapmer with all phosphorothioate internucleoside linkages;ISIS 651900 is the same as ISIS 440762, except that it included aGalNAc₃-1 at its 3′-end.

TABLE 17 Modified ASO targeting ApoC III and SRB-1 SEQ CalCd Observed IDASO Sequence (5′ to 3′) Target Mass Mass No. ISIS A_(es)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) ApoC7165.4 7164.4 4878 304801 III ISIS A_(es)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds)^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(eo) A _(do′) - ApoC 9239.5 9237.84879 647535 GalNAc ₃ -1 _(a) III ISIS A_(es)G_(eo)^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(eo)T_(eo)T_(es)A_(es)T_(eo) A_(do′) - ApoC 9142.9 9140.8 4879 647536 GalNAc ₃ -1 _(a) III ISIS T_(ks)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) SRB-1 4647.0 4646.4 4880 440762 ISIST_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(ko) A _(do′) -GalNAc ₃ -1 _(a) SRB-16721.1 6719.4 4881 651900Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicatesβ-D-2′-deoxyribonucleoside; “k” indicates 6′-(S)—CH₃ bicyclic nucleoside(e.g. cEt); “s” indicates phosphorothioate internucleoside linkages(PS); “o” indicates phosphodiester internucleoside linkages (PO); and“o′” indicates —O—P(═O)(OH)—. Superscript “m” indicates5-methylcytosines. “GalNAc₃-1” indicates a conjugate group having thestructure shown previously in Example 9. Note that GalNAc₃-1 comprises acleavable adenosine which links the ASO to remainder of the conjugate,which is designated “GalNAc₃-1_(a).” This nomenclature is used in theabove table to show the full nucleobase sequence, including theadenosine, which is part of the conjugate. Thus, in the above table, thesequences could also be listed as ending with “GalNAc₃-1” with the “Ado”omitted. This convention of using the subscript “a” to indicate theportion of a conjugate group lacking a cleavable nucleoside or cleavablemoiety is used throughout these Examples. This portion of a conjugategroup lacking the cleavable moiety is referred to herein as a “cluster”or “conjugate cluster” or “GalNAc₃ cluster.” In certain instances it isconvenient to describe a conjugate group by separately providing itscluster and its cleavable moiety.

Example 20: Dose-Dependent Antisense Inhibition of Human ApoC III inhuApoC III Transgenic Mice

ISIS 304801 and ISIS 647535, each targeting human ApoC III and describedabove, were separately tested and evaluated in a dose-dependent studyfor their ability to inhibit human ApoC III in human ApoC III transgenicmice.

Treatment

Human ApoCIII transgenic mice were maintained on a 12-hour light/darkcycle and fed ad libitum Teklad lab chow. Animals were acclimated for atleast 7 days in the research facility before initiation of theexperiment. ASOs were prepared in PBS and sterilized by filteringthrough a 0.2 micron filter. ASOs were dissolved in 0.9% PBS forinjection.

Human ApoC III transgenic mice were injected intraperitoneally once aweek for two weeks with ISIS 304801 or 647535 at 0.08, 0.25, 0.75, 2.25or 6.75 μmol/kg or with PBS as a control. Each treatment group consistedof 4 animals. Forty-eight hours after the administration of the lastdose, blood was drawn from each mouse and the mice were sacrificed andtissues were collected.

ApoC III mRNA Analysis

ApoC III mRNA levels in the mice's livers were determined usingreal-time PCR and RIBOGREEN® RNA quantification reagent (MolecularProbes, Inc. Eugene, Oreg.) according to standard protocols. ApoC IIImRNA levels were determined relative to total RNA (using Ribogreen),prior to normalization to PBS-treated control. The results below arepresented as the average percent of ApoC III mRNA levels for eachtreatment group, normalized to PBS-treated control and are denoted as “%PBS”. The half maximal effective dosage (ED₅₀) of each ASO is alsopresented in Table 18, below.

As illustrated, both antisense compounds reduced ApoC III RNA relativeto the PBS control. Further, the antisense compound conjugated toGalNAc₃-1 (ISIS 647535) was substantially more potent than the antisensecompound lacking the GalNAc₃-1 conjugate (ISIS 304801).

TABLE 18 Effect of ASO treatment on ApoC III mRNA levels in human ApoCIII transgenic mice Dose % ED₅₀ 3′ Internucleoside SEQ ASO (μmol/kg) PBS(μmol/kg) Conjugate linkage/Length ID No. PBS 0 100 — — — ISIS 0.08 950.77 None PS/20 4878 304801 0.75 42 2.25 32 6.75 19 ISIS 0.08 50 0.074GalNAc₃-1 PS/20 4879 647535 0.75 15 2.25 17 6.75 8

ApoC III Protein Analysis (Turbidometric Assay)

Plasma ApoC III protein analysis was determined using proceduresreported by Graham et al, Circulation Research, published online beforeprint Mar. 29, 2013.

Approximately 100 μl of plasma isolated from mice was analyzed withoutdilution using an Olympus Clinical Analyzer and a commercially availableturbidometric ApoC III assay (Kamiya, Cat# KAI-006, Kamiya Biomedical,Seattle, Wash.). The assay protocol was performed as described by thevendor.

As shown in the Table 19 below, both antisense compounds reduced ApoCIII protein relative to the PBS control. Further, the antisense compoundconjugated to GalNAc₃-1 (ISIS 647535) was substantially more potent thanthe antisense compound lacking the GalNAc₃-1 conjugate (ISIS 304801).

TABLE 19 Effect of ASO treatment on ApoC III plasma protein levels inhuman ApoC III transgenic mice Dose % ED₅₀ 3′ Internucleoside SEQ ASO(μmol/kg) PBS (μmol/kg) Conjugate Linkage/Length ID No. PBS 0 100 — — —ISIS 0.08 86 0.73 None PS/20 4878 304801 0.75 51 2.25 23 6.75 13 ISIS0.08 72 0.19 GalNAc₃-1 PS/20 4879 647535 0.75 14 2.25 12 6.75 11

Plasma triglycerides and cholesterol were extracted by the method ofBligh and Dyer (Bligh, E. G. and Dyer, W. J. Can. J. Biochem. Physiol.37: 911-917, 1959)(Bligh, E and Dyer, W, Can J Biochem Physiol, 37,911-917, 1959)(Bligh, E and Dyer, W, Can J Biochem Physiol, 37, 911-917,1959) and measured by using a Beckmann Coulter clinical analyzer andcommercially available reagents.

The triglyceride levels were measured relative to PBS injected mice andare denoted as “% PBS”. Results are presented in Table 20. Asillustrated, both antisense compounds lowered triglyceride levels.Further, the antisense compound conjugated to GalNAc₃-1 (ISIS 647535)was substantially more potent than the antisense compound lacking theGalNAc₃-1 conjugate (ISIS 304801).

TABLE 20 Effect of ASO treatment on triglyceride levels in transgenicmice Dose % ED₅₀ 3′ Internucleoside SEQ ASO (μmol/kg) PBS (μmol/kg)Conjugate Linkage/Length ID No. PBS 0 100 — — — ISIS 0.08 87 0.63 NonePS/20 4878 304801 0.75 46 2.25 21 6.75 12 ISIS 0.08 65 0.13 GalNAc₃-1PS/20 4879 647535 0.75 9 2.25 8 6.75 9

Plasma samples were analyzed by HPLC to determine the amount of totalcholesterol and of different fractions of cholesterol (HDL and LDL).Results are presented in Tables 21 and 22. As illustrated, bothantisense compounds lowered total cholesterol levels; both lowered LDL;and both raised HDL. Further, the antisense compound conjugated toGalNAc₃-1 (ISIS 647535) was substantially more potent than the antisensecompound lacking the GalNAc₃-1 conjugate (ISIS 304801). An increase inHDL and a decrease in LDL levels is a cardiovascular beneficial effectof antisense inhibition of ApoC III.

TABLE 21 Effect of ASO treatment on total cholesterol levels intransgenic mice Total SEQ Dose Cholesterol 3′ Internucleoside ID ASO(μmol/kg) (mg/dL) Conjugate Linkage/Length No. PBS 0 257 — — ISIS 0.08226 None PS/20 4878 304801 0.75 164 2.25 110 6.75 82 ISIS 0.08 230GalNAc ₃ -1 PS/20 4879 647535 0.75 82 2.25 86 6.75 99

TABLE 22 Effect of ASO treatment on HDL and LDL cholesterol levels intransgenic mice Dose HDL LDL 3′ Internucleoside SEQ ASO (μmol/kg)(mg/dL) (mg/dL) Conjugate Linkage/Length ID No. PBS 0 17 28 — — ISIS0.08 17 23 None PS/20 4878 304801 0.75 27 12 2.25 50 4 6.75 45 2 ISIS0.08 21 21 GalNAc₃-1 PS/20 4879 647535 0.75 44 2 2.25 50 2 6.75 58 2

Pharmacokinetics Analysis (PK)

The PK of the ASOs was also evaluated. Liver and kidney samples wereminced and extracted using standard protocols. Samples were analyzed onMSD1 utilizing IP-HPLC-MS. The tissue level (μg/g) of full-length ISIS304801 and 647535 was measured and the results are provided in Table 23.As illustrated, liver concentrations of total full-length antisensecompounds were similar for the two antisense compounds. Thus, eventhough the GalNAc₃-1-conjugated antisense compound is more active in theliver (as demonstrated by the RNA and protein data above), it is notpresent at substantially higher concentration in the liver. Indeed, thecalculated EC₅₀ (provided in Table 23) confirms that the observedincrease in potency of the conjugated compound cannot be entirelyattributed to increased accumulation. This result suggests that theconjugate improved potency by a mechanism other than liver accumulationalone, possibly by improving the productive uptake of the antisensecompound into cells.

The results also show that the concentration of GalNAc₃-1 conjugatedantisense compound in the kidney is lower than that of antisensecompound lacking the GalNAc conjugate. This has several beneficialtherapeutic implications. For therapeutic indications where activity inthe kidney is not sought, exposure to kidney risks kidney toxicitywithout corresponding benefit. Moreover, high concentration in kidneytypically results in loss of compound to the urine resulting in fasterclearance. Accordingly, for non-kidney targets, kidney accumulation isundesired. These data suggest that GalNAc₃-1 conjugation reduces kidneyaccumulation.

TABLE 23 PK analysis of ASO treatment in transgenic mice Dose LiverKidney Liver EC₅₀ 3′ Internucleoside SEQ ASO (μmol/kg) (μg/g) (μg/g)(μg/g) Conjugate Linkage/Length ID No. ISIS 0.1 5.2 2.1 53 None PS/204878 304801 0.8 62.8 119.6 2.3 142.3 191.5 6.8 202.3 337.7 ISIS 0.1 3.80.7 3.8 GalNAc₃-1 PS/20 4879 647535 0.8 72.7 34.3 2.3 106.8 111.4 6.8237.2 179.3

Metabolites of ISIS 647535 were also identified and their masses wereconfirmed by high resolution mass spectrometry analysis. The cleavagesites and structures of the observed metabolites are shown below. Therelative % of full length ASO was calculated using standard proceduresand the results are presented in Table 23a. The major metabolite of ISIS647535 was full-length ASO lacking the entire conjugate (i.e. ISIS304801), which results from cleavage at cleavage site A, shown below.Further, additional metabolites resulting from other cleavage sites werealso observed. These results suggest that introducing other cleabablebonds such as esters, peptides, disulfides, phosphoramidates oracyl-hydrazones between the GalNAc₃-1 sugar and the ASO, which can becleaved by enzymes inside the cell, or which may cleave in the reductiveenvironment of the cytosol, or which are labile to the acidic pH insideendosomes and lyzosomes, can also be useful.

TABLE 23a Observed full length metabolites of ISIS 647535 Metabolite ASOCleavage site Relative % 1 ISIS 304801 A 36.1 2 ISIS 304801 + dA B 10.53 ISIS 647535 minus [3 GalNAc] C 16.1 4 ISIS 647535 minus D 17.6 [3GalNAc + 1 5-hydroxy-pentanoic acid tether] 5 ISIS 647535 minus D 9.9 [2GalNAc + 2 5-hydroxy-pentanoic acid tether] 6 ISIS 647535 minus D 9.8 [3GalNAc + 3 5-hydroxy-pentanoic acid tether]

 

 

 

 

 

 

Example 21: Antisense Inhibition of Human ApoC III in Human ApoC IIITransgenic Mice in Single Administration Study

ISIS 304801, 647535 and 647536 each targeting human ApoC III anddescribed in Table 17, were further evaluated in a single administrationstudy for their ability to inhibit human ApoC III in human ApoC IIItransgenic mice.

Treatment

Human ApoCIII transgenic mice were maintained on a 12-hour light/darkcycle and fed ad libitum Teklad lab chow. Animals were acclimated for atleast 7 days in the research facility before initiation of theexperiment. ASOs were prepared in PBS and sterilized by filteringthrough a 0.2 micron filter. ASOs were dissolved in 0.9% PBS forinjection.

Human ApoC III transgenic mice were injected intraperitoneally once atthe dosage shown below with ISIS 304801, 647535 or 647536 (describedabove) or with PBS treated control. The treatment group consisted of 3animals and the control group consisted of 4 animals. Prior to thetreatment as well as after the last dose, blood was drawn from eachmouse and plasma samples were analyzed. The mice were sacrificed 72hours following the last administration.

Samples were collected and analyzed to determine the ApoC III mRNA andprotein levels in the liver; plasma triglycerides; and cholesterol,including HDL and LDL fractions were assessed as described above(Example 20). Data from those analyses are presented in Tables 24-28,below. Liver transaminase levels, alanine aminotransferase (ALT) andaspartate aminotransferase (AST), in serum were measured relative tosaline injected mice using standard protocols. The ALT and AST levelsshowed that the antisense compounds were well tolerated at alladministered doses.

These results show improvement in potency for antisense compoundscomprising a GalNAc₃-1 conjugate at the 3′ terminus (ISIS 647535 and647536) compared to the antisense compound lacking a GalNAc₃-1 conjugate(ISIS 304801). Further, ISIS 647536, which comprises a GalNAc₃-1conjugate and some phosphodiester linkages was as potent as ISIS 647535,which comprises the same conjugate and all internucleoside linkageswithin the ASO are phosphorothioate.

TABLE 24 Effect of ASO treatment on ApoC III mRNA levels in human ApoCIII transgenic mice Dose ED₅₀ 3′ Internucleoside SEQ ASO (mg/kg) % PBS(mg/kg) Conjugate linkage/Length ID No. PBS 0 99 — — — ISIS 1 104 13.2None PS/20 4878 304801 3 92 10 71 30 40 ISIS 0.3 98 1.9 GalNAc₃-1 PS/204879 647535 1 70 3 33 10 20 ISIS 0.3 103 1.7 GalNAc₃-1 PS/PO/20 4879647536 1 60 3 31 10 21

TABLE 25 Effect of ASO treatment on ApoC III plasma protein levels inhuman ApoC III transgenic mice Dose ED₅₀ 3′ Internucleoside SEQ ASO(mg/kg) % PBS (mg/kg) Conjugate Linkage/Length ID No. PBS 0 99 — — —ISIS 1 104 23.2 None PS/20 4878 304801 3 92 10 71 30 40 ISIS 0.3 98 2.1GalNAc₃-1 PS/20 4879 647535 1 70 3 33 10 20 ISIS 0.3 103 1.8 GalNAc₃-1PS/PO/20 4879 647536 1 60 3 31 10 21

TABLE 26 Effect of ASO treatment on triglyceride levels in transgenicmice Dose ED₅₀ 3′ Internucleoside SEQ ASO (mg/kg) % PBS (mg/kg)Conjugate Linkage/Length ID No. PBS 0 98 — — — ISIS 1 80 29.1 None PS/204878 304801 3 92 10 70 30 47 ISIS 0.3 100 2.2 GalNAc₃-1 PS/20 4879647535 1 70 3 34 10 23 ISIS 0.3 95 1.9 GalNAc₃-1 PS/PO/20 4879 647536 166 3 31 10 23

TABLE 27 Effect of ASO treatment on total cholesterol levels intransgenic mice Dose 3′ Internucleoside SEQ ASO (mg/kg) % PBS ConjugateLinkage/Length ID No. PBS 0 96 — — ISIS 1 104 None PS/20 4878 304801 396 10 86 30 72 ISIS 0.3 93 GalNAc₃-1 PS/20 4879 647535 1 85 3 61 10 53ISIS 0.3 115 GalNAc₃-1 PS/PO/20 4879 647536 1 79 3 51 10 54

TABLE 28 Effect of ASO treatment on HDL and LDL cholesterol levels intransgenic mice Dose HDL LDL 3′ Internucleoside SEQ ASO (mg/kg) % PBS %PBS Conjugate Linkage/Length ID No. PBS 0 131 90 — — ISIS 1 130 72 NonePS/20 4878 304801 3 186 79 10 226 63 30 240 46 ISIS 0.3 98 86 GalNAc₃-1PS/20 4879 647535 1 214 67 3 212 39 10 218 35 ISIS 0.3 143 89 GalNAc₃-1PS/PO/20 4879 647536 1 187 56 3 213 33 10 221 34

These results confirm that the GalNAc₃-1 conjugate improves potency ofan antisense compound. The results also show equal potency of aGalNAc₃-1 conjugated antisense compounds where the antisenseoligonucleotides have mixed linkages (ISIS 647536 which has sixphosphodiester linkages) and a full phosphorothioate version of the sameantisense compound (ISIS 647535).

Phosphorothioate linkages provide several properties to antisensecompounds. For example, they resist nuclease digestion and they bindproteins resulting in accumulation of compound in the liver, rather thanin the kidney/urine. These are desirable properties, particularly whentreating an indication in the liver. However, phosphorothioate linkageshave also been associated with an inflammatory response. Accordingly,reducing the number of phosphorothioate linkages in a compound isexpected to reduce the risk of inflammation, but also lowerconcentration of the compound in liver, increase concentration in thekidney and urine, decrease stability in the presence of nucleases, andlower overall potency. The present results show that a GalNAc₃-1conjugated antisense compound where certain phosphorothioate linkageshave been replaced with phosphodiester linkages is as potent against atarget in the liver as a counterpart having full phosphorothioatelinkages. Such compounds are expected to be less proinflammatory (SeeExample 24 describing an experiment showing reduction of PS results inreduced inflammatory effect).

Example 22: Effect of GalNAc₃-1 Conjugated Modified ASO Targeting SRB-1In Vivo

ISIS 440762 and 651900, each targeting SRB-1 and described in Table 17,were evaluated in a dose-dependent study for their ability to inhibitSRB-1 in Balb/c mice.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected subcutaneously once at the dosage shown below with ISIS 440762,651900 or with PBS treated control. Each treatment group consisted of 4animals. The mice were sacrificed 48 hours following the finaladministration to determine the SRB-1 mRNA levels in liver usingreal-time PCR and RIBOGREEN® RNA quantification reagent (MolecularProbes, Inc. Eugene, Oreg.) according to standard protocols. SRB-1 mRNAlevels were determined relative to total RNA (using Ribogreen), prior tonormalization to PBS-treated control. The results below are presented asthe average percent of SRB-1 mRNA levels for each treatment group,normalized to PBS-treated control and is denoted as “% PBS”.

As illustrated in Table 29, both antisense compounds lowered SRB-1 mRNAlevels. Further, the antisense compound comprising the GalNAc₃-1conjugate (ISIS 651900) was substantially more potent than the antisensecompound lacking the GalNAc₃-1 conjugate (ISIS 440762). These resultsdemonstrate that the potency benefit of GalNAc₃-1 conjugates areobserved using antisense oligonucleotides complementary to a differenttarget and having different chemically modified nucleosides, in thisinstance modified nucleosides comprise constrained ethyl sugar moieties(a bicyclic sugar moiety).

TABLE 29 Effect of ASO treatment on SRB-1 mRNA levels in Balb/c miceLiver SEQ Dose % ED₅₀ 3′ Internucleoside ID ASO (mg/kg) PBS (mg/kg)Conjugate linkage/Length No. PBS 0 100 — — ISIS 0.7 85 2.2 None PS/144880 440762 2 55 7 12 20 3 ISIS 0.07 98 0.3 GalNAc₃-1 PS/14 4881 6519000.2 63 0.7 20 2 6 7 5

Example 23: Human Peripheral Blood Mononuclear Cells (hPBMC) AssayProtocol

The hPBMC assay was performed using BD Vautainer CPT tube method. Asample of whole blood from volunteered donors with informed consent atUS HealthWorks clinic (Faraday & El Camino Real, Carlsbad) was obtainedand collected in 4-15 BD Vacutainer CPT 8 ml tubes (VWR Cat.# BD362753).The approximate starting total whole blood volume in the CPT tubes foreach donor was recorded using the PBMC assay data sheet.

The blood sample was remixed immediately prior to centrifugation bygently inverting tubes 8-10 times. CPT tubes were centrifuged at rt(18-25° C.) in a horizontal (swing-out) rotor for 30 min. at 1500-1800RCF with brake off (2700 RPM Beckman Allegra 6R). The cells wereretrieved from the buffy coat interface (between Ficoll and polymer gellayers); transferred to a sterile 50 ml conical tube and pooled up to 5CPT tubes/50 ml conical tube/donor. The cells were then washed twicewith PBS (Ca⁺⁺, Mg⁺⁺ free; GIBCO). The tubes were topped up to 50 ml andmixed by inverting several times. The sample was then centrifuged at330×g for 15 minutes at rt (1215 RPM in Beckman Allegra 6R) andaspirated as much supernatant as possible without disturbing pellet. Thecell pellet was dislodged by gently swirling tube and resuspended cellsin RPMI+10% FBS+pen/strep (˜1 ml/10 ml starting whole blood volume). A60 μl sample was pipette into a sample vial (Beckman Coulter) with 600μl VersaLyse reagent (Beckman Coulter Cat# A09777) and was gentlyvortexed for 10-15 sec. The sample was allowed to incubate for 10 min.at rt and being mixed again before counting. The cell suspension wascounted on Vicell XR cell viability analyzer (Beckman Coulter) usingPBMC cell type (dilution factor of 1:11 was stored with otherparameters). The live cell/ml and viability were recorded. The cellsuspension was diluted to 1×10⁷ live PBMC/ml in RPMI+10% FBS+pen/strep.

The cells were plated at 5×10⁵ in 50 l/well of 96-well tissue cultureplate (Falcon Microtest). 50 μl/well of 2× concentration oligos/controlsdiluted in RPMI+10% FBS+pen/strep. was added according to experimenttemplate (100 μl/well total). Plates were placed on the shaker andallowed to mix for approx. 1 min. After being incubated for 24 hrs at37° C.; 5% CO₂, the plates were centrifuged at 400×g for 10 minutesbefore removing the supernatant for MSD cytokine assay (i.e. human IL-6,IL-10, IL-8 and MCP-1).

Example 24: Evaluation of Proinflammatory Effects in hPBMC Assay forGalNAc₃-1 Conjugated ASOs

The antisense oligonucleotides (ASOs) listed in Table 30 were evaluatedfor proinflammatory effect in hPBMC assay using the protocol describedin Example 23. ISIS 353512 is an internal standard known to be a highresponder for IL-6 release in the assay. The hPBMCs were isolated fromfresh, volunteered donors and were treated with ASOs at 0, 0.0128,0.064, 0.32, 1.6, 8, 40 and 200 μM concentrations. After a 24 hrtreatment, the cytokine levels were measured.

The levels of IL-6 were used as the primary readout. The EC₅₀ and Em,was calculated using standard procedures. Results are expressed as theaverage ratio of E_(max)/EC₅₀ from two donors and is denoted as“E_(max)/EC₅₀.” The lower ratio indicates a relative decrease in theproinflammatory response and the higher ratio indicates a relativeincrease in the proinflammatory response.

With regard to the test compounds, the least proinflammatory compoundwas the PS/PO linked ASO (ISIS 616468). The GalNAc₃-1 conjugated ASO,ISIS 647535 was slightly less proinflammatory than its non-conjugatedcounterpart ISIS 304801. These results indicate that incorporation ofsome PO linkages reduces proinflammatory reaction and addition of aGalNAc₃-1 conjugate does not make a compound more proinflammatory andmay reduce proinflammatory response. Accordingly, one would expect thatan antisense compound comprising both mixed PS/PO linkages and aGalNAc₃-1 conjugate would produce lower proinflammatory responsesrelative to full PS linked antisense compound with or without aGalNAc₃-1 conjugate. These results show that GalNAc₃-1 conjugatedantisense compounds, particularly those having reduced PS content areless proinflammatory.

Together, these results suggest that a GalNAc₃-1 conjugated compound,particularly one with reduced PS content, can be administered at ahigher dose than a counterpart full PS antisense compound lacking aGalNAc₃-1 conjugate. Since half-life is not expected to be substantiallydifferent for these compounds, such higher administration would resultin less frequent dosing. Indeed such administration could be even lessfrequent, because the GalNAc₃-1 conjugated compounds are more potent(See Examples 20-22) and re-dosing is necessary once the concentrationof a compound has dropped below a desired level, where such desiredlevel is based on potency.

TABLE 30 Modified ASOs SEQ ID ASO Sequence (5′ to 3′) Target No. ISISG_(es) ^(m)C_(es)T_(es)G_(es)A_(es)T_(ds)T_(ds)A_(ds)G_(ds)A_(ds)G_(ds)TNFα 4882 104838 A_(ds)G_(ds)A_(ds)G_(ds)G_(es)T_(es) ^(m)C_(es)^(m)C_(es) ^(m)C_(e) ISIS T_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(ds)A_(ds)T_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds) CRP 4883353512 G_(ds)A_(ds)G_(ds)A_(ds) ^(m)C_(ds) ^(m)C_(ds)T_(es)G_(es)G_(e)ISIS A_(es)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ApoC III 4878 304801 ^(m)C_(ds)^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) ISISA_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds)ApoC III 4879 647535 ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds)^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a) ISISA_(es)G_(eo) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds)ApoC III 4878 616468 ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds)^(m)C_(ds)T_(eo)T_(eo)T_(es)A_(es)T_(e) Subscripts: “e” indicates 2′-MOEmodified nucleoside; “d” indicates β-D-2′-deoxyribonucleoside;“k” indicates 6′-(S)—CH₃ bicyclic nucleoside (e.g. cEt); “s” indicatesphosphorothioate internucleoside linkages (PS); “o” indicatesphosphodiester internucleoside linkages (PO); and “o′” indicates—O—P(═O)(OH)—. Superscript “m” indicates 5-methylcytosines.“A_(do′)-GalNAc₃-1_(a)” indicates a conjugate having the structureGalNAc₃-1 shown in Example 9 attached to the 3′-end of the antisenseoligonucleotide, as indicated.

TABLE 31 Proinflammatory Effect of ASOs targeting ApoC III in hPBMCassay SEQ EC₅₀ E_(max) 3′ Internucleoside ID ASO (μM) (μM) E_(max)/EC₅₀Conjugate Linkage/Length No. ISIS 0.01 265.9 26,590 None PS/20 4883353512 (high responder) ISIS 0.07 106.55 1,522 None PS/20 4878 304801ISIS 0.12 138 1,150 GalNAc₃-1 PS/20 4879 647535 ISIS 0.32 71.52 224 NonePS/PO/20 4878 616468

Example 25: Effect of GalNAc₃-1 Conjugated Modified ASO Targeting HumanApoC III In Vitro

ISIS 304801 and 647535 described above were tested in vitro. Primaryhepatocyte cells from transgenic mice at a density of 25,000 cells perwell were treated with 0.03, 0.08, 0.24, 0.74, 2.22, 6.67 and 20 μMconcentrations of modified oligonucleotides. After a treatment period ofapproximately 16 hours, RNA was isolated from the cells and mRNA levelswere measured by quantitative real-time PCR and the hApoC III mRNAlevels were adjusted according to total RNA content, as measured byRIBOGREEN.

The IC₅₀ was calculated using the standard methods and the results arepresented in Table 32. As illustrated, comparable potency was observedin cells treated with ISIS 647535 as compared to the control, ISIS304801.

TABLE 32 Modified ASO targeting human ApoC III in primary hepatocytesInternucleoside SEQ ASO IC₅₀ (μM) 3′ Conjugate linkage/Length ID No.ISIS 0.44 None PS/20 4878 304801 ISIS 0.31 GalNAc ₃ -1 PS/20 4879 647535

In this experiment, the large potency benefits of GalNAc₃-1 conjugationthat are observed in vivo were not observed in vitro. Subsequent freeuptake experiments in primary hepatocytes in vitro did show increasedpotency of oligonucleotides comprising various GalNAc conjugatesrelative to oligonucleotides that lacking the GalNAc conjugate. (seeExamples 60, 82, and 92)

Example 26: Effect of PO/PS Linkages on ApoC III ASO Activity

Human ApoC III transgenic mice were injected intraperitoneally once at25 mg/kg of ISIS 304801, or ISIS 616468 (both described above) or withPBS treated control once per week for two weeks. The treatment groupconsisted of 3 animals and the control group consisted of 4 animals.Prior to the treatment as well as after the last dose, blood was drawnfrom each mouse and plasma samples were analyzed. The mice weresacrificed 72 hours following the last administration.

Samples were collected and analyzed to determine the ApoC III proteinlevels in the liver as described above (Example 20). Data from thoseanalyses are presented in Table 33, below.

These results show reduction in potency for antisense compounds withPO/PS (ISIS 616468) in the wings relative to full PS (ISIS 304801).

TABLE 33 Effect of ASO treatment on ApoC III protein levels in humanApoC III transgenic mice Dose 3′ Internucleoside SEQ ID ASO (mg/kg) %PBS Conjugate linkage/Length No. PBS 0 99 — — ISIS 25 mg/kg/wk 24 NoneFull PS 4878 304801 for 2 wks ISIS 25 mg/kg/wk 40 None 14 PS/6 PO 4878616468 for 2 wks

Example 27: Compound 56

Compound 56 is commercially available from Glen Research or may beprepared according to published procedures reported by Shchepinov etal., Nucleic Acids Research, 1997, 25(22), 4447-4454.

Example 28: Preparation of Compound 60

Compound 4 was prepared as per the procedures illustrated in Example 2.Compound 57 is commercially available. Compound 60 was confirmed bystructural analysis.

Compound 57 is meant to be representative and not intended to belimiting as other monoprotected substituted or unsubstituted alkyl diolsincluding but not limited to those presented in the specification hereincan be used to prepare phosphoramidites having a predeterminedcomposition.

Example 29: Preparation of Compound 63

Compounds 61 and 62 are prepared using procedures similar to thosereported by Tober et al., Eur. J Org. Chem., 2013, 3, 566-577; and Jianget al., Tetrahedron, 2007, 63(19), 3982-3988.

Alternatively, Compound 63 is prepared using procedures similar to thosereported in scientific and patent literature by Kim et al., Synlett,2003, 12, 1838-1840; and Kim et al., published PCT InternationalApplication, WO 2004063208.

Example 30: Preparation of Compound 63b

Compound 63a is prepared using procedures similar to those reported byHanessian et al., Canadian Journal of Chemistry, 1996, 74(9), 1731-1737.

Example 31: Preparation of Compound 63d

Compound 63c is prepared using procedures similar to those reported byChen et al., Chinese Chemical Letters, 1998, 9(5), 451-453.

Example 32: Preparation of Compound 67

Compound 64 was prepared as per the procedures illustrated in Example 2.Compound 65 is prepared using procedures similar to those reported by Oret al., published PCT International Application, WO 2009/003009. Theprotecting groups used for Compound 65 are meant to be representativeand not intended to be limiting as other protecting groups including butnot limited to those presented in the specification herein can be used.

Example 33: Preparation of Compound 70

Compound 64 was prepared as per the procedures illustrated in Example 2.Compound 68 is commercially available. The protecting group used forCompound 68 is meant to be representative and not intended to belimiting as other protecting groups including but not limited to thosepresented in the specification herein can be used.

Example 34: Preparation of Compound 75a

Compound 75 is prepared according to published procedures reported byShchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454.

Example 35: Preparation of Compound 79

Compound 76 was prepared according to published procedures reported byShchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454.

Example 36: Preparation of Compound 79a

Compound 77 is prepared as per the procedures illustrated in Example 35.

Example 37: General Method for the Preparation of Conjugated OligomericCompound 82 Comprising a Phosphodiester Linked GalNAc₃-2 Conjugate at 5′Terminus Via Solid Support (Method I)

wherein GalNAc₃-2 has the structure:

The GalNAc₃ cluster portion of the conjugate group GalNAc₃-2(GalNAc₃-2_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. Wherein GalNAc₃-2_(a) has the formula:

The VIMAD-bound oligomeric compound 79b was prepared using standardprocedures for automated DNA/RNA synthesis (see Dupouy et al., Angew.Chem. Int. Ed., 2006, 45, 3623-3627). The phosphoramidite Compounds 56and 60 were prepared as per the procedures illustrated in Examples 27and 28, respectively. The phosphoramidites illustrated are meant to berepresentative and not intended to be limiting as other phosphoramiditebuilding blocks including but not limited those presented in thespecification herein can be used to prepare an oligomeric compoundhaving a phosphodiester linked conjugate group at the 5′ terminus. Theorder and quantity of phosphoramidites added to the solid support can beadjusted to prepare the oligomeric compounds as described herein havingany predetermined sequence and composition.

Example 38: Alternative Method for the Preparation of OligomericCompound 82 Comprising a Phosphodiester Linked GalNAc₃-2 Conjugate at 5′Terminus (Method II)

The VIMAD-bound oligomeric compound 79b was prepared using standardprocedures for automated DNA/RNA synthesis (see Dupouy et al., Angew.Chem. Int. Ed., 2006, 45, 3623-3627). The GalNAc₃-2 clusterphosphoramidite, Compound 79 was prepared as per the proceduresillustrated in Example 35. This alternative method allows a one-stepinstallation of the phosphodiester linked GalNAc₃-2 conjugate to theoligomeric compound at the final step of the synthesis. Thephosphoramidites illustrated are meant to be representative and notintended to be limiting, as other phosphoramidite building blocksincluding but not limited to those presented in the specification hereincan be used to prepare oligomeric compounds having a phosphodiesterconjugate at the 5′ terminus. The order and quantity of phosphoramiditesadded to the solid support can be adjusted to prepare the oligomericcompounds as described herein having any predetermined sequence andcomposition.

Example 39: General Method for the Preparation of Oligomeric Compound83h Comprising a GalNAc₃-3 Conjugate at the 5′ Terminus (GalNAc₃-1Modified for 5′ End Attachment) Via Solid Support

Compound 18 was prepared as per the procedures illustrated in Example 4.Compounds 83a and 83b are commercially available. Oligomeric Compound83e comprising a phosphodiester linked hexylamine was prepared usingstandard oligonucleotide synthesis procedures. Treatment of theprotected oligomeric compound with aqueous ammonia provided the5′-GalNAc₃-3 conjugated oligomeric compound (83h).

Wherein GalNAc₃-3 has the structure:

The GalNAc₃ cluster portion of the conjugate group GalNAc₃-3(GalNAc₃-3_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. Wherein GalNAc₃-3_(a) has the formula:

Example 40: General Method for the Preparation of Oligomeric Compound 89Comprising a Phosphodiester Linked GalNAc₃-4 Conjugate at the 3′Terminus Via Solid Support

Wherein GalNAc₃-4 has the structure:

Wherein CM is a cleavable moiety. In certain embodiments, cleavablemoiety is:

The GalNAc₃ cluster portion of the conjugate group GalNAc₃-4(GalNAc₃-4_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. Wherein GalNAc₃-4_(a) has the formula:

The protected Unylinker functionalized solid support Compound 30 iscommercially available. Compound 84 is prepared using procedures similarto those reported in the literature (see Shchepinov et al., NucleicAcids Research, 1997, 25(22), 4447-4454; Shchepinov et al., NucleicAcids Research, 1999, 27, 3035-3041; and Hornet et al., Nucleic AcidsResearch, 1997, 25, 4842-4849).

The phosphoramidite building blocks, Compounds 60 and 79a are preparedas per the procedures illustrated in Examples 28 and 36. Thephosphoramidites illustrated are meant to be representative and notintended to be limiting as other phosphoramidite building blocks can beused to prepare an oligomeric compound having a phosphodiester linkedconjugate at the 3′ terminus with a predetermined sequence andcomposition. The order and quantity of phosphoramidites added to thesolid support can be adjusted to prepare the oligomeric compounds asdescribed herein having any predetermined sequence and composition.

Example 41: General Method for the Preparation of ASOs Comprising aPhosphodiester Linked GalNAc₃-2 (See Example 37, Bx is Adenine)Conjugate at the 5′ Position Via Solid Phase Techniques (Preparation ofISIS 661134)

Unless otherwise stated, all reagents and solutions used for thesynthesis of oligomeric compounds are purchased from commercial sources.Standard phosphoramidite building blocks and solid support are used forincorporation nucleoside residues which include for example T, A, G, and^(m)C residues. Phosphoramidite compounds 56 and 60 were used tosynthesize the phosphodiester linked GalNAc₃-2 conjugate at the 5′terminus. A 0.1 μM solution of phosphoramidite in anhydrous acetonitrilewas used for 3-D-2′-deoxyribonucleoside and 2′-MOE.

The ASO syntheses were performed on ABI 394 synthesizer (1-2 μmol scale)or on GE Healthcare Bioscience ÄKTA oligopilot synthesizer (40-200 μmolscale) by the phosphoramidite coupling method on VIMAD solid support(110 μmol/g, Guzaev et al., 2003) packed in the column. For the couplingstep, the phosphoramidites were delivered at a 4 fold excess over theinitial loading of the solid support and phosphoramidite coupling wascarried out for 10 min. All other steps followed standard protocolssupplied by the manufacturer. A solution of 6% dichloroacetic acid intoluene was used for removing the dimethoxytrityl (DMT) groups from5′-hydroxyl groups of the nucleotide. 4,5-Dicyanoimidazole (0.7 M) inanhydrous CH₃CN was used as activator during the coupling step.Phosphorothioate linkages were introduced by sulfurization with 0.1 μMsolution of xanthane hydride in 1:1 pyridine/CH₃CN for a contact time of3 minutes. A solution of 20% tert-butylhydroperoxide in CH₃CN containing6% water was used as an oxidizing agent to provide phosphodiesterinternucleoside linkages with a contact time of 12 minutes.

After the desired sequence was assembled, the cyanoethyl phosphateprotecting groups were deprotected using a 20% diethylamine in toluene(v/v) with a contact time of 45 minutes. The solid-support bound ASOswere suspended in aqueous ammonia (28-30 wt %) and heated at 55° C. for6 h. The unbound ASOs were then filtered and the ammonia was boiled off.The residue was purified by high pressure liquid chromatography on astrong anion exchange column (GE Healthcare Bioscience, Source 30Q, 30μm, 2.54×8 cm, A=100 mM ammonium acetate in 30% aqueous CH₃CN, B=1.5 μMNaBr in A, 0-40% of B in 60 min, flow 14 mL min-1, =260 nm). The residuewas desalted by HPLC on a reverse phase column to yield the desired ASOsin an isolated yield of 15-30% based on the initial loading on the solidsupport. The ASOs were characterized by ion-pair-HPLC coupled MSanalysis with Agilent 1100 MSD system.

TABLE 34 ASO comprising a phosphodiester linked GalNAc₃-2 conjugate atthe 5′ position targeting SRB-1 Observed SEQ ID ISIS No. Sequence (5′ to3′) CalCd Mass Mass No. 661134 GalNAc ₃ -2 _(a) - _(o′) A _(do)T_(ks)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) 6482.2 6481.6 4884G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) Subscripts: “e” indicates2′-MOE modified nucleoside; “d” indicates β-D-2′-deoxyribonucleoside;“k” indicates 6′-(S)—CH₃ bicyclic nucleoside (e.g. cEt); “s” indicatesphosphorothioate internucleoside linkages (PS); “o” indicatesphosphodiester internucleoside linkages (PO); and “o′” indicates—O—P(═O)(OH)—. Superscript “m” indicates 5-methylcytosines. Thestructure of GalNAc₃-2_(a) is shown in Example 37.

Example 42: General Method for the Preparation of ASOs Comprising aGalNAc₃-3 Conjugate at the 5′ Position Via Solid Phase Techniques(Preparation of ISIS 661166)

The synthesis for ISIS 661166 was performed using similar procedures asillustrated in Examples 39 and 41.

ISIS 661166 is a 5-10-5 MOE gapmer, wherein the 5′ position comprises aGalNAc₃-3 conjugate. The ASO was characterized by ion-pair-HPLC coupledMS analysis with Agilent 1100 MSD system.

TABLE 34A ASO comprising a GalNAc₃-3 conjugate at the 5′ position via ahexylamino phosphodiester linkage targeting Malat-1 ISIS Calcd ObservedNo. Sequence (5′ to 3′) Conjugate Mass Mass SEQ ID No. 661166 5′-GalNAc₃ -3 _(a-o′) ^(m)C_(es)G_(es)G_(es)T_(es)G_(es) 5′ -GalNAc ₃ -3 8992.168990.51 4885 ^(m)C_(ds)A_(ds)A_(ds)G_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(ds)A_(ds)G_(ds) G_(es)A_(es)A_(es)T_(es)T_(e)Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicatesβ-D-2′-deoxyribonucleoside; “s” indicates phosphorothioateinternucleoside linkages (PS); “o” indicates phosphodiesterinternucleoside linkages (PO); and “o′” indicates —O—P(═O)(OH)—.Superscript “m” indicates 5-methylcytosines. The structure of“5′-GalNAc₃-3a” is shown in Example 39.

Example 43: Dose-Dependent Study of Phosphodiester Linked GalNAc₃-2 (SeeExamples 37 and 41, Bx is Adenine) at the 5′ Terminus Targeting SRB-1 InVivo

ISIS 661134 (see Example 41) comprising a phosphodiester linkedGalNAc₃-2 conjugate at the 5′ terminus was tested in a dose-dependentstudy for antisense inhibition of SRB-1 in mice. Unconjugated ISIS440762 and 651900 (GalNAc₃-1 conjugate at 3′ terminus, see Example 9)were included in the study for comparison and are described previouslyin Table 17.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected subcutaneously once at the dosage shown below with ISIS 440762,651900, 661134 or with PBS treated control. Each treatment groupconsisted of 4 animals. The mice were sacrificed 72 hours following thefinal administration to determine the liver SRB-1 mRNA levels usingreal-time PCR and RIBOGREEN® RNA quantification reagent (MolecularProbes, Inc. Eugene, Oreg.) according to standard protocols. SRB-1 mRNAlevels were determined relative to total RNA (using Ribogreen), prior tonormalization to PBS-treated control. The results below are presented asthe average percent of SRB-1 mRNA levels for each treatment group,normalized to PBS-treated control and is denoted as “% PBS”. The ED₅₀swere measured using similar methods as described previously and arepresented below.

As illustrated in Table 35, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner. Indeed, theantisense oligonucleotides comprising the phosphodiester linkedGalNAc₃-2 conjugate at the 5′ terminus (ISIS 661134) or the GalNAc₃-1conjugate linked at the 3′ terminus (ISIS 651900) showed substantialimprovement in potency compared to the unconjugated antisenseoligonucleotide (ISIS 440762). Further, ISIS 661134, which comprises thephosphodiester linked GalNAc₃-2 conjugate at the 5′ terminus wasequipotent compared to ISIS 651900, which comprises the GalNAc₃-1conjugate at the 3′ terminus.

TABLE 35 ASOs containing GalNAc₃-1 or GalNAc₃-2 targeting SRB-1 ISISDosage SRB-1 mRNA ED₅₀ SEQ No. (mg/kg) levels (% PBS) (mg/kg) ConjugateID No. PBS 0 100 — — 440762 0.2 116 2.58 No conjugate 4880 0.7 91 2 69 722 20 5 651900 0.07 95 0.26 3′ GalNAc ₃ -1 4881 0.2 77 0.7 28 2 11 7 8661134 0.07 107 0.25 5′ GalNAc ₃ -2 4881 0.2 86 0.7 28 2 10 7 6

Structures for 3′ GalNAc₃-1 and 5′ GalNAc₃-2 were described previouslyin Examples 9 and 37.

Pharmacokinetics Analysis (PK)

The PK of the ASOs from the high dose group (7 mg/kg) was examined andevaluated in the same manner as illustrated in Example 20. Liver samplewas minced and extracted using standard protocols. The full lengthmetabolites of 661134 (5′ GalNAc₃-2) and ISIS 651900 (3′ GalNAc₃-1) wereidentified and their masses were confirmed by high resolution massspectrometry analysis. The results showed that the major metabolitedetected for the ASO comprising a phosphodiester linked GalNAc₃-2conjugate at the 5′ terminus (ISIS 661134) was ISIS 440762 (data notshown). No additional metabolites, at a detectable level, were observed.Unlike its counterpart, additional metabolites similar to those reportedpreviously in Table 23a were observed for the ASO having the GalNAc₃-1conjugate at the 3′ terminus (ISIS 651900). These results suggest thathaving the phosphodiester linked GalNAc₃-1 or GalNAc₃-2 conjugate mayimprove the PK profile of ASOs without compromising their potency.

Example 44: Effect of PO/PS Linkages on Antisense Inhibition of ASOsComprising GalNAc₃-1 Conjugate (See Example 9) at the 3′ TerminusTargeting SRB-1

ISIS 655861 and 655862 comprising a GalNAc₃-1 conjugate at the 3′terminus each targeting SRB-1 were tested in a single administrationstudy for their ability to inhibit SRB-1 in mice. The parentunconjugated compound, ISIS 353382 was included in the study forcomparison.

The ASOs are 5-10-5 MOE gapmers, wherein the gap region comprises ten2′-deoxyribonucleosides and each wing region comprises five 2′-MOEmodified nucleosides. The ASOs were prepared using similar methods asillustrated previously in Example 19 and are described Table 36, below.

TABLE 36 Modified ASOs comprising GalNAc₃-1 conjugate at the 3′ terminustargeting SRB-1 SEQ ID ISIS No. Sequence (5′ to 3′) Chemistry No. 353382G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) Full PS no conjugate 4886 (parent)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 655861 G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) Full PS with 4887^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′)-GalNAc ₃ -1 _(a) GalNAc ₃ -1 conjugate 655862 G_(es)^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) Mixed PS/PO with 4887^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(eo) A _(do′)-GalNAc ₃ -1a GalNAc ₃ -1 conjugate Subscripts: “e” indicates 2′-MOEmodified nucleoside; “d” indicates β-D-2′-deoxyribonucleoside;“s” indicates phosphorothioate internucleoside linkages (PS);“o” indicates phosphodiester internucleoside linkages (PO); and“o′” indicates —O—P(═O)(OH)—. Superscript “m” indicates5-methylcytosines. The structure of “GalNAc₃-1” is shown in Example 9.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected subcutaneously once at the dosage shown below with ISIS 353382,655861, 655862 or with PBS treated control. Each treatment groupconsisted of 4 animals. Prior to the treatment as well as after the lastdose, blood was drawn from each mouse and plasma samples were analyzed.The mice were sacrificed 72 hours following the final administration todetermine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN®RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.)according to standard protocols. SRB-1 mRNA levels were determinedrelative to total RNA (using Ribogreen), prior to normalization toPBS-treated control. The results below are presented as the averagepercent of SRB-1 mRNA levels for each treatment group, normalized toPBS-treated control and is denoted as “% PBS”. The ED₅₀s were measuredusing similar methods as described previously and are reported below.

As illustrated in Table 37, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner compared to PBStreated control. Indeed, the antisense oligonucleotides comprising theGalNAc₃-1 conjugate at the 3′ terminus (ISIS 655861 and 655862) showedsubstantial improvement in potency comparing to the unconjugatedantisense oligonucleotide (ISIS 353382). Further, ISIS 655862 with mixedPS/PO linkages showed an improvement in potency relative to full PS(ISIS 655861).

TABLE 37 Effect of PO/PS linkages on antisense inhibition of ASOscomprising GalNAc₃-1 conjugate at 3′ terminus targeting SRB-1 ISISDosage SRB-1 mRNA ED₅₀ SEQ No. (mg/kg) levels (% PBS) (mg/kg) ChemistryID No. PBS 0 100 — — 353382 3 76.65 10.4 Full PS without 4886 (parent)10 52.40 conjugate 30 24.95 655861 0.5 81.22 2.2 Full PS with 4887 1.563.51 GalNAc₃-1 5 24.61 conjugate 15 14.80 655862 0.5 69.57 1.3 MixedPS/PO 4887 1.5 45.78 with 5 19.70 GalNAc₃-1 15 12.90 conjugate

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were measured relative to salineinjected mice using standard protocols. Organ weights were alsoevaluated. The results demonstrated that no elevation in transaminaselevels (Table 38) or organ weights (data not shown) were observed inmice treated with ASOs compared to PBS control. Further, the ASO withmixed PS/PO linkages (ISIS 655862) showed similar transaminase levelscompared to full PS (ISIS 655861).

TABLE 38 Effect of PO/PS linkages on transaminase levels of ASOscomprising GalNAc₃-1 conjugate at 3′ terminus targeting SRB-1 Dosage ALTAST SEQ ISIS No. (mg/kg) (U/L) (U/L) Chemistry ID No. PBS 0 28.5 65 —353382 3 50.25 89 Full PS without 4886 (parent) 10 27.5 79.3 conjugate30 27.3 97 655861 0.5 28 55.7 Full PS with 4887 1.5 30 78 GalNAc₃-1 5 2963.5 15 28.8 67.8 655862 0.5 50 75.5 Mixed PS/PO with 4887 1.5 21.7 58.5GalNAc₃-1 5 29.3 69 15 22 61

Example 45: Preparation of PFP Ester, Compound 110a

Compound 4 (9.5 g, 28.8 mmoles) was treated with compound 103a or 103b(38 mmoles), individually, and TMSOTf (0.5 eq.) and molecular sieves indichloromethane (200 mL), and stirred for 16 hours at room temperature.At that time, the organic layer was filtered thru celite, then washedwith sodium bicarbonate, water and brine. The organic layer was thenseparated and dried over sodium sulfate, filtered and reduced underreduced pressure. The resultant oil was purified by silica gelchromatography (2%->10% methanol/dichloromethane) to give compounds 104aand 104b in >80% yield. LCMS and proton NMR was consistent with thestructure.

Compounds 104a and 104b were treated to the same conditions as forcompounds 100a-d (Example 47), to give compounds 105a and 105b in >90%yield. LCMS and proton NMR was consistent with the structure.

Compounds 105a and 105b were treated, individually, with compound 90under the same conditions as for compounds 901a-d, to give compounds106a (80%) and 106b (20%). LCMS and proton NMR was consistent with thestructure.

Compounds 106a and 106b were treated to the same conditions as forcompounds 96a-d (Example 47), to give 107a (60%) and 107b (20%). LCMSand proton NMR was consistent with the structure.

Compounds 107a and 107b were treated to the same conditions as forcompounds 97a-d (Example 47), to give compounds 108a and 108b in 40-60%yield. LCMS and proton NMR was consistent with the structure.

Compounds 108a (60%) and 108b (40%) were treated to the same conditionsas for compounds 100a-d (Example 47), to give compounds 109a and 109bin >80% yields. LCMS and proton NMR was consistent with the structure.

Compound 109a was treated to the same conditions as for compounds 101a-d(Example 47), to give Compound 110a in 30-60% yield. LCMS and proton NMRwas consistent with the structure. Alternatively, Compound 110b can beprepared in a similar manner starting with Compound 109b.

Example 46: General Procedure for Conjugation with PFP Esters(Oligonucleotide 111); Preparation of ISIS 666881 (GalNAc₃-10)

A 5′-hexylamino modified oligonucleotide was synthesized and purifiedusing standard solid-phase oligonucleotide procedures. The 5′-hexylaminomodified oligonucleotide was dissolved in 0.1 μM sodium tetraborate, pH8.5 (200 μL) and 3 equivalents of a selected PFP esterified GalNAc₃cluster dissolved in DMSO (50 μL) was added. If the PFP esterprecipitated upon addition to the ASO solution DMSO was added until allPFP ester was in solution. The reaction was complete after about 16 h ofmixing at room temperature. The resulting solution was diluted withwater to 12 mL and then spun down at 3000 rpm in a spin filter with amass cut off of 3000 Da. This process was repeated twice to remove smallmolecule impurities. The solution was then lyophilized to dryness andredissolved in concentrated aqueous ammonia and mixed at roomtemperature for 2.5 h followed by concentration in vacuo to remove mostof the ammonia. The conjugated oligonucleotide was purified and desaltedby RP-HPLC and lyophilized to provide the GalNAc₃ conjugatedoligonucleotide.

Oligonucleotide 111 is conjugated with GalNAc₃-10. The GalNAc₃ clusterportion of the conjugate group GalNAc₃-10 (GalNAc₃-10_(a)) can becombined with any cleavable moiety to provide a variety of conjugategroups. In certain embodiments, the cleavable moiety is—P(═O)(OH)-A_(d)-P(═O)(OH)— as shown in the oligonucleotide (ISIS666881) synthesized with GalNAc₃-10 below. The structure of GalNAc₃-10(GalNAc₃-10_(a)-CM-) is shown below:

Following this general procedure ISIS 666881 was prepared. 5′-hexylaminomodified oligonucleotide, ISIS 660254, was synthesized and purifiedusing standard solid-phase oligonucleotide procedures. ISIS 660254 (40mg, 5.2 μmol) was dissolved in 0.1 μM sodium tetraborate, pH 8.5 (200μL) and 3 equivalents PFP ester (Compound 110a) dissolved in DMSO (50μL) was added. The PFP ester precipitated upon addition to the ASOsolution requiring additional DMSO (600 μL) to fully dissolve the PFPester. The reaction was complete after 16 h of mixing at roomtemperature. The solution was diluted with water to 12 mL total volumeand spun down at 3000 rpm in a spin filter with a mass cut off of 3000Da. This process was repeated twice to remove small molecule impurities.The solution was lyophilized to dryness and redissolved in concentratedaqueous ammonia with mixing at room temperature for 2.5 h followed byconcentration in vacuo to remove most of the ammonia. The conjugatedoligonucleotide was purified and desalted by RP-HPLC and lyophilized togive ISIS 666881 in 90% yield by weight (42 mg, 4.7 μmol).

GalNAc₃-10 conjugated oligonucleotide SEQ ASO Sequence (5′ to 3′)5′ group ID No. ISIS 660254 NH₂(CH₂)₆-_(o)A_(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) Hexylamine 4888^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(es)T_(ds) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) ISIS 666881 GalNAc ₃ -10 _(a) - _(o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) GalNAc ₃-10 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es)^(m)C_(es) ^(m)C_(es)T_(es)T_(e) Capital letters indicate the nucleobasefor each nucleoside and ^(m)C indicates a 5-methyl cytosine. Subscripts:“e” indicates a 2′-MOE modified nucleoside; “d” indicates aβ-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioateinternucleoside linkage (PS); “o” indicates a phosphodiesterinternucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—.Conjugate groups are in bold.

Example 47: Preparation of Oligonucleotide 102 Comprising GalNAc₃-8

The triacid 90 (4 g, 14.43 mmol) was dissolved in DMF (120 mL) andN,N-Diisopropylethylamine (12.35 mL, 72 mmoles). Pentafluorophenyltrifluoroacetate (8.9 mL, 52 mmoles) was added dropwise, under argon,and the reaction was allowed to stir at room temperature for 30 minutes.Boc-diamine 91a or 91b (68.87 mmol) was added, along withN,N-Diisopropylethylamine (12.35 mL, 72 mmoles), and the reaction wasallowed to stir at room temperature for 16 hours. At that time, the DMFwas reduced by >75% under reduced pressure, and then the mixture wasdissolved in dichloromethane. The organic layer was washed with sodiumbicarbonate, water and brine. The organic layer was then separated anddried over sodium sulfate, filtered and reduced to an oil under reducedpressure. The resultant oil was purified by silica gel chromatography(2%->10% methanol/dichloromethane) to give compounds 92a and 92b in anapproximate 80% yield. LCMS and proton NMR were consistent with thestructure.

Compound 92a or 92b (6.7 mmoles) was treated with 20 mL ofdichloromethane and 20 mL of trifluoroacetic acid at room temperaturefor 16 hours. The resultant solution was evaporated and then dissolvedin methanol and treated with DOWEX-OH resin for 30 minutes. Theresultant solution was filtered and reduced to an oil under reducedpressure to give 85-90% yield of compounds 93a and 93b.

Compounds 7 or 64 (9.6 mmoles) were treated with HBTU (3.7 g, 9.6mmoles) and N,N-Diisopropylethylamine (5 mL) in DMF (20 mL) for 15minutes. To this was added either compounds 93a or 93b (3 mmoles), andallowed to stir at room temperature for 16 hours. At that time, the DMFwas reduced by >75% under reduced pressure, and then the mixture wasdissolved in dichloromethane. The organic layer was washed with sodiumbicarbonate, water and brine. The organic layer was then separated anddried over sodium sulfate, filtered and reduced to an oil under reducedpressure. The resultant oil was purified by silica gel chromatography(5%->20% methanol/dichloromethane) to give compounds 96a-d in 20-40%yield. LCMS and proton NMR was consistent with the structure.

Compounds 96a-d (0.75 mmoles), individually, were hydrogenated overRaney Nickel for 3 hours in Ethanol (75 mL). At that time, the catalystwas removed by filtration thru celite, and the ethanol removed underreduced pressure to give compounds 97a-d in 80-90% yield. LCMS andproton NMR were consistent with the structure.

Compound 23 (0.32 g, 0.53 mmoles) was treated with HBTU (0.2 g, 0.53mmoles) and N,N-Diisopropylethylamine (0.19 mL, 1.14 mmoles) in DMF (30mL) for 15 minutes. To this was added compounds 97a-d (0.38 mmoles),individually, and allowed to stir at room temperature for 16 hours. Atthat time, the DMF was reduced by >75% under reduced pressure, and thenthe mixture was dissolved in dichloromethane. The organic layer waswashed with sodium bicarbonate, water and brine. The organic layer wasthen separated and dried over sodium sulfate, filtered and reduced to anoil under reduced pressure. The resultant oil was purified by silica gelchromatography (2%->20% methanol/dichloromethane) to give compounds98a-d in 30-40% yield. LCMS and proton NMR was consistent with thestructure.

Compound 99 (0.17 g, 0.76 mmoles) was treated with HBTU (0.29 g, 0.76mmoles) and N,N-Diisopropylethylamine (0.35 mL, 2.0 mmoles) in DMF (50mL) for 15 minutes. To this was added compounds 97a-d (0.51 mmoles),individually, and allowed to stir at room temperature for 16 hours. Atthat time, the DMF was reduced by >75% under reduced pressure, and thenthe mixture was dissolved in dichloromethane. The organic layer waswashed with sodium bicarbonate, water and brine. The organic layer wasthen separated and dried over sodium sulfate, filtered and reduced to anoil under reduced pressure. The resultant oil was purified by silica gelchromatography (5%->20% methanol/dichloromethane) to give compounds100a-d in 40-60% yield. LCMS and proton NMR was consistent with thestructure.

Compounds 100a-d (0.16 mmoles), individually, were hydrogenated over 10%Pd(OH)₂/C for 3 hours in methanol/ethyl acetate (1:1, 50 mL). At thattime, the catalyst was removed by filtration thru celite, and theorganics removed under reduced pressure to give compounds 101a-d in80-90% yield. LCMS and proton NMR was consistent with the structure.

Compounds 101a-d (0.15 mmoles), individually, were dissolved in DMF (15mL) and pyridine (0.016 mL, 0.2 mmoles). Pentafluorophenyltrifluoroacetate (0.034 mL, 0.2 mmoles) was added dropwise, under argon,and the reaction was allowed to stir at room temperature for 30 minutes.At that time, the DMF was reduced by >75% under reduced pressure, andthen the mixture was dissolved in dichloromethane. The organic layer waswashed with sodium bicarbonate, water and brine. The organic layer wasthen separated and dried over sodium sulfate, filtered and reduced to anoil under reduced pressure. The resultant oil was purified by silica gelchromatography (2%->5% methanol/dichloromethane) to give compounds102a-d in an approximate 80% yield. LCMS and proton NMR were consistentwith the structure.

Oligomeric Compound 102, comprising a GalNAc₃-8 conjugate group, wasprepared using the general procedures illustrated in Example 46. TheGalNAc₃ cluster portion of the conjugate group GalNAc₃-8 (GalNAc₃-8_(a))can be combined with any cleavable moiety to provide a variety ofconjugate groups. In a preferred embodiment, the cleavable moiety is—P(═O)(OH)-A_(d)-P(═O)(OH)—.

The structure of GalNAc₃-8 (GalNAc₃-8_(a)-CM-) is shown below:

Example 48: Preparation of Oligonucleotide 119 Comprising GalNAc₃-7

Compound 112 was synthesized following the procedure described in theliterature (J. Med. Chem. 2004, 47, 5798-5808).

Compound 112 (5 g, 8.6 mmol) was dissolved in 1:1 methanol/ethyl acetate(22 mL/22 mL). Palladium hydroxide on carbon (0.5 g) was added. Thereaction mixture was stirred at room temperature under hydrogen for 12h. The reaction mixture was filtered through a pad of celite and washedthe pad with 1:1 methanol/ethyl acetate. The filtrate and the washingswere combined and concentrated to dryness to yield Compound 105a(quantitative). The structure was confirmed by LCMS.

Compound 113 (1.25 g, 2.7 mmol), HBTU (3.2 g, 8.4 mmol) and DIEA (2.8mL, 16.2 mmol) were dissolved in anhydrous DMF (17 mL) and the reactionmixture was stirred at room temperature for 5 min. To this a solution ofCompound 105a (3.77 g, 8.4 mmol) in anhydrous DMF (20 mL) was added. Thereaction was stirred at room temperature for 6 h. Solvent was removedunder reduced pressure to get an oil. The residue was dissolved inCH₂Cl₂ (100 mL) and washed with aqueous saturated NaHCO₃ solution (100mL) and brine (100 mL). The organic phase was separated, dried (Na₂SO₄),filtered and evaporated. The residue was purified by silica gel columnchromatography and eluted with 10 to 20% MeOH in dichloromethane toyield Compound 114 (1.45 g, 30%). The structure was confirmed by LCMSand ¹H NMR analysis.

Compound 114 (1.43 g, 0.8 mmol) was dissolved in 1:1 methanol/ethylacetate (4 mL/4 mL). Palladium on carbon (wet, 0.14 g) was added. Thereaction mixture was flushed with hydrogen and stirred at roomtemperature under hydrogen for 12 h. The reaction mixture was filteredthrough a pad of celite. The celite pad was washed with methanol/ethylacetate (1:1). The filtrate and the washings were combined together andevaporated under reduced pressure to yield Compound 115 (quantitative).The structure was confirmed by LCMS and ¹H NMR analysis.

Compound 83a (0.17 g, 0.75 mmol), HBTU (0.31 g, 0.83 mmol) and DIEA(0.26 mL, 1.5 mmol) were dissolved in anhydrous DMF (5 mL) and thereaction mixture was stirred at room temperature for 5 min. To this asolution of Compound 115 (1.22 g, 0.75 mmol) in anhydrous DMF was addedand the reaction was stirred at room temperature for 6 h. The solventwas removed under reduced pressure and the residue was dissolved inCH₂Cl₂. The organic layer was washed aqueous saturated NaHCO₃ solutionand brine and dried over anhydrous Na₂SO₄ and filtered. The organiclayer was concentrated to dryness and the residue obtained was purifiedby silica gel column chromatography and eluted with 3 to 15% MeOH indichloromethane to yield Compound 116 (0.84 g, 61%). The structure wasconfirmed by LC MS and ¹H NMR analysis.

Compound 116 (0.74 g, 0.4 mmol) was dissolved in 1:1 methanol/ethylacetate (5 mL/5 mL). Palladium on carbon (wet, 0.074 g) was added. Thereaction mixture was flushed with hydrogen and stirred at roomtemperature under hydrogen for 12 h. The reaction mixture was filteredthrough a pad of celite. The celite pad was washed with methanol/ethylacetate (1:1). The filtrate and the washings were combined together andevaporated under reduced pressure to yield compound 117 (0.73 g, 98%).The structure was confirmed by LCMS and ¹H NMR analysis.

Compound 117 (0.63 g, 0.36 mmol) was dissolved in anhydrous DMF (3 mL).To this solution N,N-Diisopropylethylamine (70 μL, 0.4 mmol) andpentafluorophenyl trifluoroacetate (72 μL, 0.42 mmol) were added. Thereaction mixture was stirred at room temperature for 12 h and pouredinto a aqueous saturated NaHCO₃ solution. The mixture was extracted withdichloromethane, washed with brine and dried over anhydrous Na₂SO₄. Thedichloromethane solution was concentrated to dryness and purified withsilica gel column chromatography and eluted with 5 to 10% MeOH indichloromethane to yield compound 118 (0.51 g, 79%). The structure wasconfirmed by LCMS and ¹H and ¹H and ¹⁹F NMR.

Oligomeric Compound 119, comprising a GalNAc₃-7 conjugate group, wasprepared using the general procedures illustrated in Example 46. TheGalNAc₃ cluster portion of the conjugate group GalNAc₃-7 (GalNAc₃-7_(a))can be combined with any cleavable moiety to provide a variety ofconjugate groups. In certain embodiments, the cleavable moiety is—P(═O)(OH)-A_(d)-P(═O)(OH)—.

The structure of GalNAc₃-7 (GalNAc₃-7_(a)-CM-) is shown below:

Example 49: Preparation of Oligonucleotide 132 Comprising GalNAc₃-5

Compound 120 (14.01 g, 40 mmol) and HBTU (14.06 g, 37 mmol) weredissolved in anhydrous DMF (80 mL). Triethylamine (11.2 mL, 80.35 mmol)was added and stirred for 5 min. The reaction mixture was cooled in anice bath and a solution of compound 121 (10 g, mmol) in anhydrous DMF(20 mL) was added. Additional triethylamine (4.5 mL, 32.28 mmol) wasadded and the reaction mixture was stirred for 18 h under an argonatmosphere. The reaction was monitored by TLC (ethyl acetate:hexane;1:1; Rf=0.47). The solvent was removed under reduced pressure. Theresidue was taken up in EtOAc (300 mL) and washed with 1M NaHSO₄ (3×150mL), aqueous saturated NaHCO₃ solution (3×150 mL) and brine (2×100 mL).Organic layer was dried with Na₂SO₄. Drying agent was removed byfiltration and organic layer was concentrated by rotary evaporation.Crude mixture was purified by silica gel column chromatography andeluted by using 35-50% EtOAc in hexane to yield a compound 122 (15.50 g,78.13%). The structure was confirmed by LCMS and ¹H NMR analysis. Massm/z 589.3 [M+H]⁺.

A solution of LiOH (92.15 mmol) in water (20 mL) and THF (10 mL) wasadded to a cooled solution of Compound 122 (7.75 g, 13.16 mmol)dissolved in methanol (15 mL). The reaction mixture was stirred at roomtemperature for 45 min. and monitored by TLC (EtOAc:hexane; 1:1). Thereaction mixture was concentrated to half the volume under reducedpressure. The remaining solution was cooled an ice bath and neutralizedby adding concentrated HCl. The reaction mixture was diluted, extractedwith EtOAc (120 mL) and washed with brine (100 mL). An emulsion formedand cleared upon standing overnight. The organic layer was separateddried (Na₂SO₄), filtered and evaporated to yield Compound 123 (8.42 g).Residual salt is the likely cause of excess mass. LCMS is consistentwith structure. Product was used without any further purification.M.W.cal:574.36; M.W.fd:575.3 [M+H]⁺.

Compound 126 was synthesized following the procedure described in theliterature (J. Am. Chem. Soc. 2011, 133, 958-963).

Compound 123 (7.419 g, 12.91 mmol), HOBt (3.49 g, 25.82 mmol) andcompound 126 (6.33 g, 16.14 mmol) were dissolved in and DMF (40 mL) andthe resulting reaction mixture was cooled in an ice bath. To thisN,N-Diisopropylethylamine (4.42 mL, 25.82 mmol), PyBop (8.7 g, 16.7mmol) followed by Bop coupling reagent (1.17 g, 2.66 mmol) were addedunder an argon atmosphere. The ice bath was removed and the solution wasallowed to warm to room temperature. The reaction was completed after 1h as determined by TLC (DCM:MeOH:AA; 89:10:1). The reaction mixture wasconcentrated under reduced pressure. The residue was dissolved in EtOAc(200 mL) and washed with 1 μM NaHSO₄ (3×100 mL), aqueous saturatedNaHCO₃ (3×100 mL) and brine (2×100 mL). The organic phase separateddried (Na₂SO₄), filtered and concentrated. The residue was purified bysilica gel column chromatography with a gradient of 50% hexanes/EtOAC to100% EtOAc to yield Compound 127 (9.4 g) as a white foam. LCMS and ¹HNMR were consistent with structure. Mass m/z 778.4 [M+H]⁺.

Trifluoroacetic acid (12 mL) was added to a solution of compound 127(1.57 g, 2.02 mmol) in dichloromethane (12 mL) and stirred at roomtemperature for 1 h. The reaction mixture was co-evaporated with toluene(30 mL) under reduced pressure to dryness. The residue obtained wasco-evaporated twice with acetonitrile (30 mL) and toluene (40 mL) toyield Compound 128 (1.67 g) as trifluoro acetate salt and used for nextstep without further purification. LCMS and ¹H NMR were consistent withstructure. Mass m/z 478.2 [M+H]⁺.

Compound 7 (0.43 g, 0.963 mmol), HATU (0.35 g, 0.91 mmol), and HOAt(0.035 g, 0.26 mmol) were combined together and dried for 4 h over P₂O₅under reduced pressure in a round bottom flask and then dissolved inanhydrous DMF (1 mL) and stirred for 5 min. To this a solution ofcompound 128 (0.20 g, 0.26 mmol) in anhydrous DMF (0.2 mL) andN,N-Diisopropylethylamine (0.2 mL) was added. The reaction mixture wasstirred at room temperature under an argon atmosphere. The reaction wascomplete after 30 min as determined by LCMS and TLC (7% MeOH/DCM). Thereaction mixture was concentrated under reduced pressure. The residuewas dissolved in DCM (30 mL) and washed with 1 μM NaHSO₄ (3×20 mL),aqueous saturated NaHCO₃ (3×20 mL) and brine (3×20 mL). The organicphase was separated, dried over Na₂SO₄, filtered and concentrated. Theresidue was purified by silica gel column chromatography using 5-15%MeOH in dichloromethane to yield Compound 129 (96.6 mg). LC MS and ¹HNMR are consistent with structure. Mass m/z 883.4 [M+2H]⁺.

Compound 129 (0.09 g, 0.051 mmol) was dissolved in methanol (5 mL) in 20mL scintillation vial. To this was added a small amount of 10% Pd/C(0.015 mg) and the reaction vessel was flushed with H₂ gas. The reactionmixture was stirred at room temperature under H₂ atmosphere for 18 h.The reaction mixture was filtered through a pad of Celite and the Celitepad was washed with methanol. The filtrate washings were pooled togetherand concentrated under reduced pressure to yield Compound 130 (0.08 g).LCMS and ¹H NMR were consistent with structure. The product was usedwithout further purification. Mass m/z 838.3 [M+2H]⁺.

To a 10 mL pointed round bottom flask were added compound 130 (75.8 mg,0.046 mmol), 0.37 M pyridine/DMF (200 μL) and a stir bar. To thissolution was added 0.7 μM pentafluorophenyl trifluoroacetate/DMF (100μL) drop wise with stirring. The reaction was completed after 1 h asdetermined by LC MS. The solvent was removed under reduced pressure andthe residue was dissolved in CHCl₃ (˜10 mL).

The organic layer was partitioned against NaHSO₄ (1 μM, 10 mL), aqueoussaturated NaHCO₃ (10 mL) and brine (10 mL) three times each. The organicphase separated and dried over Na₂SO₄, filtered and concentrated toyield Compound 131 (77.7 mg). LCMS is consistent with structure. Usedwithout further purification. Mass m/z 921.3 [M+2H]⁺.

Oligomeric Compound 132, comprising a GalNAc₃-5 conjugate group, wasprepared using the general procedures illustrated in Example 46. TheGalNAc₃ cluster portion of the conjugate group GalNAc₃-5 (GalNAc₃-5_(a))can be combined with any cleavable moiety to provide a variety ofconjugate groups. In certain embodiments, the cleavable moiety is—P(═O)(OH)-A_(d)-P(═O)(OH)—.

The structure of GalNAc₃-5 (GalNAc₃-5_(a)-CM-) is shown below:

Example 50: Preparation of Oligonucleotide 144 Comprising GalNAc₄-11

Synthesis of Compound 134. To a Merrifield flask was added aminomethylVIMAD resin (2.5 g, 450 mol/g) that was washed with acetonitrile,dimethylformamide, dichloromethane and acetonitrile. The resin wasswelled in acetonitrile (4 mL). Compound 133 was pre-activated in a 100mL round bottom flask by adding 20 (1.0 mmol, 0.747 g), TBTU (1.0 mmol,0.321 g), acetonitrile (5 mL) and DIEA (3.0 mmol, 0.5 mL). This solutionwas allowed to stir for 5 min and was then added to the Merrifield flaskwith shaking. The suspension was allowed to shake for 3 h. The reactionmixture was drained and the resin was washed with acetonitrile, DMF andDCM. New resin loading was quantitated by measuring the absorbance ofthe DMT cation at 500 nm (extinction coefficient=76000) in DCM anddetermined to be 238 μmol/g. The resin was capped by suspending in anacetic anhydride solution for ten minutes three times.

The solid support bound compound 141 was synthesized using iterativeFmoc-based solid phase peptide synthesis methods. A small amount ofsolid support was withdrawn and suspended in aqueous ammonia (28-30 wt%) for 6 h. The cleaved compound was analyzed by LC-MS and the observedmass was consistent with structure. Mass m/z 1063.8 [M+2H]⁺.

The solid support bound compound 142 was synthesized using solid phasepeptide synthesis methods.

The solid support bound compound 143 was synthesized using standardsolid phase synthesis on a DNA synthesizer.

The solid support bound compound 143 was suspended in aqueous ammonia(28-30 wt %) and heated at 55° C. for 16 h. The solution was cooled andthe solid support was filtered. The filtrate was concentrated and theresidue dissolved in water and purified by HPLC on a strong anionexchange column. The fractions containing full length compound 144 werepooled together and desalted. The resulting GalNAc₄-11 conjugatedoligomeric compound was analyzed by LC-MS and the observed mass wasconsistent with structure.

The GalNAc₄ cluster portion of the conjugate group GalNAc₄-11(GalNAc₄-11_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. In certain embodiments, the cleavablemoiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—.

The structure of GalNAc₄-11 (GalNAc₄-11_(a)-CM) is shown below:

Example 51: Preparation of Oligonucleotide 155 Comprising GalNAc₃-6

Compound 146 was synthesized as described in the literature (AnalyticalBiochemistry 1995, 229, 54-60).

Compound 4 (15 g, 45.55 mmol) and compound 35b (14.3 grams, 57 mmol)were dissolved in CH₂Cl₂ (200 ml). Activated molecular sieves (4 Å. 2 g,powdered) were added, and the reaction was allowed to stir for 30minutes under nitrogen atmosphere. TMS-OTf was added (4.1 ml, 22.77mmol) and the reaction was allowed to stir at room temp overnight. Uponcompletion, the reaction was quenched by pouring into solution ofsaturated aqueous NaHCO₃ (500 ml) and crushed ice (˜150 g). The organiclayer was separated, washed with brine, dried over MgSO₄, filtered, andwas concentrated to an orange oil under reduced pressure. The crudematerial was purified by silica gel column chromatography and elutedwith 2-10% MeOH in CH₂Cl₂ to yield Compound 112 (16.53 g, 63%). LCMS and¹H NMR were consistent with the expected compound.

Compound 112 (4.27 g, 7.35 mmol) was dissolved in 1:1 MeOH/EtOAc (40ml). The reaction mixture was purged by bubbling a stream of argonthrough the solution for 15 minutes. Pearlman's catalyst (palladiumhydroxide on carbon, 400 mg) was added, and hydrogen gas was bubbledthrough the solution for 30 minutes.

Upon completion (TLC 10% MeOH in CH₂Cl₂, and LCMS), the catalyst wasremoved by filtration through a pad of celite. The filtrate wasconcentrated by rotary evaporation, and was dried briefly under highvacuum to yield Compound 105a (3.28 g). LCMS and 1H NMR were consistentwith desired product.

Compound 147 (2.31 g, 11 mmol) was dissolved in anhydrous DMF (100 mL).N,N-Diisopropylethylamine (DIEA, 3.9 mL, 22 mmol) was added, followed byHBTU (4 g, 10.5 mmol). The reaction mixture was allowed to stir for ˜15minutes under nitrogen. To this a solution of compound 105a (3.3 g, 7.4mmol) in dry DMF was added and stirred for 2 h under nitrogenatmosphere. The reaction was diluted with EtOAc and washed withsaturated aqueous NaHCO₃ and brine. The organics phase was separated,dried (MgSO₄), filtered, and concentrated to an orange syrup. The crudematerial was purified by column chromatography 2-5% MeOH in CH₂Cl₂ toyield Compound 148 (3.44 g, 73%). LCMS and ¹H NMR were consistent withthe expected product.

Compound 148 (3.3 g, 5.2 mmol) was dissolved in 1:1 MeOH/EtOAc (75 ml).The reaction mixture was purged by bubbling a stream of argon throughthe solution for 15 minutes. Pearlman's catalyst (palladium hydroxide oncarbon) was added (350 mg). Hydrogen gas was bubbled through thesolution for 30 minutes. Upon completion (TLC 10% MeOH in DCM, andLCMS), the catalyst was removed by filtration through a pad of celite.The filtrate was concentrated by rotary evaporation, and was driedbriefly under high vacuum to yield Compound 149 (2.6 g). LCMS wasconsistent with desired product. The residue was dissolved in dry DMF(10 ml) was used immediately in the next step.

Compound 146 (0.68 g, 1.73 mmol) was dissolved in dry DMF (20 ml). Tothis DIEA (450 μL, 2.6 mmol, 1.5 eq.) and HBTU (1.96 g, 0.5.2 mmol) wereadded. The reaction mixture was allowed to stir for 15 minutes at roomtemperature under nitrogen. A solution of compound 149 (2.6 g) inanhydrous DMF (10 mL) was added. The pH of the reaction was adjusted topH=9-10 by addition of DIEA (if necessary). The reaction was allowed tostir at room temperature under nitrogen for 2 h. Upon completion thereaction was diluted with EtOAc (100 mL), and washed with aqueoussaturated aqueous NaHCO₃, followed by brine. The organic phase wasseparated, dried over MgSO₄, filtered, and concentrated. The residue waspurified by silica gel column chromatography and eluted with 2-10% MeOHin CH₂Cl₂ to yield Compound 150 (0.62 g, 20%). LCMS and ¹H NMR wereconsistent with the desired product.

Compound 150 (0.62 g) was dissolved in 1:1 MeOH/EtOAc (5 L). Thereaction mixture was purged by bubbling a stream of argon through thesolution for 15 minutes. Pearlman's catalyst (palladium hydroxide oncarbon) was added (60 mg). Hydrogen gas was bubbled through the solutionfor 30 minutes. Upon completion (TLC 10% MeOH in DCM, and LCMS), thecatalyst was removed by filtration (syringe-tip Teflon filter, 0.45 μm).The filtrate was concentrated by rotary evaporation, and was driedbriefly under high vacuum to yield Compound 151 (0.57 g). The LCMS wasconsistent with the desired product. The product was dissolved in 4 mLdry DMF and was used immediately in the next step.

Compound 83a (0.11 g, 0.33 mmol) was dissolved in anhydrous DMF (5 mL)and N,N-Diisopropylethylamine (75 μL, 1 mmol) and PFP-TFA (90 μL, 0.76mmol) were added. The reaction mixture turned magenta upon contact, andgradually turned orange over the next 30 minutes. Progress of reactionwas monitored by TLC and LCMS. Upon completion (formation of the PFPester), a solution of compound 151 (0.57 g, 0.33 mmol) in DMF was added.The pH of the reaction was adjusted to pH=9-10 by addition ofN,N-Diisopropylethylamine (if necessary). The reaction mixture wasstirred under nitrogen for ˜30 min. Upon completion, the majority of thesolvent was removed under reduced pressure. The residue was diluted withCH₂Cl₂ and washed with aqueous saturated NaHCO₃, followed by brine. Theorganic phase separated, dried over MgSO₄, filtered, and concentrated toan orange syrup. The residue was purified by silica gel columnchromatography (2-10% MeOH in CH₂Cl₂) to yield Compound 152 (0.35 g,55%). LCMS and ¹H NMR were consistent with the desired product.

Compound 152 (0.35 g, 0.182 mmol) was dissolved in 1:1 MeOH/EtOAc (10mL). The reaction mixture was purged by bubbling a stream of argon thruthe solution for 15 minutes. Pearlman's catalyst (palladium hydroxide oncarbon) was added (35 mg). Hydrogen gas was bubbled thru the solutionfor 30 minutes. Upon completion (TLC 10% MeOH in DCM, and LCMS), thecatalyst was removed by filtration (syringe-tip Teflon filter, 0.45 μm).The filtrate was concentrated by rotary evaporation, and was driedbriefly under high vacuum to yield Compound 153 (0.33 g, quantitative).The LCMS was consistent with desired product.

Compound 153 (0.33 g, 0.18 mmol) was dissolved in anhydrous DMF (5 mL)with stirring under nitrogen. To this N,N-Diisopropylethylamine (65 μL,0.37 mmol) and PFP-TFA (35 μL, 0.28 mmol) were added. The reactionmixture was stirred under nitrogen for ˜30 min. The reaction mixtureturned magenta upon contact, and gradually turned orange. The pH of thereaction mixture was maintained at pH=9-10 by adding moreN,-Diisopropylethylamine. The progress of the reaction was monitored byTLC and LCMS. Upon completion, the majority of the solvent was removedunder reduced pressure. The residue was diluted with CH₂Cl₂ (50 mL), andwashed with saturated aqueous NaHCO₃, followed by brine. The organiclayer was dried over MgSO₄, filtered, and concentrated to an orangesyrup. The residue was purified by column chromatography and eluted with2-10% MeOH in CH₂Cl₂ to yield Compound 154 (0.29 g, 79%). LCMS and ¹HNMR were consistent with the desired product.

Oligomeric Compound 155, comprising a GalNAc₃-6 conjugate group, wasprepared using the general procedures illustrated in Example 46. TheGalNAc₃ cluster portion of the conjugate group GalNAc₃-6 (GalNAc₃-6_(a))can be combined with any cleavable moiety to provide a variety ofconjugate groups. In certain embodiments, the cleavable moiety is—P(═O)(OH)-A_(d)-P(═O)(OH)—.

The structure of GalNAc₃-6 (GalNAc₃-6_(a)-CM-) is shown below:

Example 52: Preparation of Oligonucleotide 160 Comprising GalNAc₃-9

Compound 156 was synthesized following the procedure described in theliterature (J. Med. Chem. 2004, 47, 5798-5808).

Compound 156, (18.60 g, 29.28 mmol) was dissolved in methanol (200 mL).Palladium on carbon (6.15 g, 10 wt %, loading (dry basis), matrix carbonpowder, wet) was added. The reaction mixture was stirred at roomtemperature under hydrogen for 18 h. The reaction mixture was filteredthrough a pad of celite and the celite pad was washed thoroughly withmethanol. The combined filtrate was washed and concentrated to dryness.The residue was purified by silica gel column chromatography and elutedwith 5-10% methanol in dichloromethane to yield Compound 157 (14.26 g,89%). Mass m/z 544.1 [M−H]⁻.

Compound 157 (5 g, 9.17 mmol) was dissolved in anhydrous DMF (30 mL).HBTU (3.65 g, 9.61 mmol) and N,N-Diisopropylethylamine (13.73 mL, 78.81mmol) were added and the reaction mixture was stirred at roomtemperature for 5 minutes. To this a solution of compound 47 (2.96 g,7.04 mmol) was added.

The reaction was stirred at room temperature for 8 h. The reactionmixture was poured into a saturated NaHCO₃ aqueous solution. The mixturewas extracted with ethyl acetate and the organic layer was washed withbrine and dried (Na₂SO₄), filtered and evaporated. The residue obtainedwas purified by silica gel column chromatography and eluted with 50%ethyl acetate in hexane to yield compound 158 (8.25 g, 73.3%). Thestructure was confirmed by MS and ¹H NMR analysis.

Compound 158 (7.2 g, 7.61 mmol) was dried over P₂O₅ under reducedpressure. The dried compound was dissolved in anhydrous DMF (50 mL). Tothis 1H-tetrazole (0.43 g, 6.09 mmol) and N-methylimidazole (0.3 mL,3.81 mmol) and 2-cyanoethyl-N,N,N′,N′-tetraisopropyl phosphorodiamidite(3.65 mL, 11.50 mmol) were added. The reaction mixture was stirred tunder an argon atmosphere for 4 h. The reaction mixture was diluted withethyl acetate (200 mL). The reaction mixture was washed with saturatedNaHCO₃ and brine. The organic phase was separated, dried (Na₂SO₄),filtered and evaporated. The residue was purified by silica gel columnchromatography and eluted with 50-90% ethyl acetate in hexane to yieldCompound 159 (7.82 g, 80.5%). The structure was confirmed by LCMS and³¹P NMR analysis.

Oligomeric Compound 160, comprising a GalNAc₃-9 conjugate group, wasprepared using standard oligonucleotide synthesis procedures. Threeunits of compound 159 were coupled to the solid support, followed bynucleotide phosphoramidites. Treatment of the protected oligomericcompound with aqueous ammonia yielded compound 160. The GalNAc₃ clusterportion of the conjugate group GalNAc₃-9 (GalNAc₃-9_(a)) can be combinedwith any cleavable moiety to provide a variety of conjugate groups. Incertain embodiments, the cleavable moiety is—P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-9(GalNAc₃-9_(a)-CM) is shown below:

Example 53: Alternate Procedure for Preparation of Compound 18(GalNAc₃-1a and GalNAc₃-3a)

Lactone 161 was reacted with diamino propane (3-5 eq) or Mono-Bocprotected diamino propane (1 eq) to provide alcohol 162a or 162b. Whenunprotected propanediamine was used for the above reaction, the excessdiamine was removed by evaporation under high vacuum and the free aminogroup in 162a was protected using CbzC1 to provide 162b as a white solidafter purification by column chromatography. Alcohol 162b was furtherreacted with compound 4 in the presence of TMSOTfto provide 163a whichwas converted to 163b by removal of the Cbz group using catalytichydrogenation. The pentafluorophenyl (PFP) ester 164 was prepared byreacting triacid 113 (see Example 48) with PFPTFA (3.5 eq) and pyridine(3.5 eq) in DMF (0.1 to 0.5 M). The triester 164 was directly reactedwith the amine 163b (3-4 eq) and DIPEA (3-4 eq) to provide Compound 18.The above method greatly facilitates purification of intermediates andminimizes the formation of byproducts which are formed using theprocedure described in Example 4.

Example 54: Alternate Procedure for Preparation of Compound 18(GalNAc₃-1a and GalNAc₃-3a)

The triPFP ester 164 was prepared from acid 113 using the procedureoutlined in example 53 above and reacted with mono-Boc protected diamineto provide 165 in essentially quantitative yield. The Boc groups wereremoved with hydrochloric acid or trifluoroacetic acid to provide thetriamine which was reacted with the PFP activated acid 166 in thepresence of a suitable base such as DIPEA to provide Compound 18.

The PFP protected Gal-NAc acid 166 was prepared from the correspondingacid by treatment with PFPTFA (1-1.2 eq) and pyridine (1-1.2 eq) in DMF.The precursor acid in turn was prepared from the corresponding alcoholby oxidation using TEMPO (0.2 eq) and BAIB in acetonitrile and water.The precursor alcohol was prepared from sugar intermediate 4 by reactionwith 1,6-hexanediol (or 1,5-pentanediol or other diol for other nvalues) (2-4 eq) and TMSOTf using conditions described previously inexample 47.

Example 55: Dose-Dependent Study of Oligonucleotides Comprising Either a3′ or 5′-Conjugate Group (Comparison of GalNAc₃-1, 3, 8 and 9) TargetingSRB-1 In Vivo

The oligonucleotides listed below were tested in a dose-dependent studyfor antisense inhibition of SRB-1 in mice. Unconjugated ISIS 353382 wasincluded as a standard. Each of the various GalNAc₃ conjugate groups wasattached at either the 3′ or 5′ terminus of the respectiveoligonucleotide by a phosphodiester linked 2′-deoxyadenosine nucleoside(cleavable moiety).

TABLE 39 Modified ASO targeting SRB-1 SEQ ID ASO Sequence (5′ to 3′)Motif Conjugate No. ISIS 353382 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5none 4886 (parent) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) ISIS 655861 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5GalNAc ₃ -1 4887 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a) ISIS 664078 G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 GalNAc ₃ -9 4887^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′)-GalNAc ₃ -9 _(a) ISIS 661161 GalNAc ₃ -3 _(a) - _(o′) A _(do) 5/10/5GalNAc ₃ -3 4888 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 665001GalNAc ₃ -8 _(a) - _(o′) A _(do) 5/10/5 GalNAc ₃ -8 4888 G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) Capital letters indicate the nucleobase for eachnucleoside and ^(m)C indicates a 5-methyl cytosine. Subscripts:“e” indicates a 2′-MOE modified nucleoside; “d” indicates aβ-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioateinternucleoside linkage (PS); “o” indicates a phosphodiesterinternucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—.Conjugate groups are in bold.

The structure of GalNAc₃-1a was shown previously in Example 9. Thestructure of GalNAc₃-9 was shown previously in Example 52. The structureof GalNAc₃-3 was shown previously in Example 39. The structure ofGalNAc₃-8 was shown previously in Example 47.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected subcutaneously once at the dosage shown below with ISIS 353382,655861, 664078, 661161, 665001 or with saline. Each treatment groupconsisted of 4 animals. The mice were sacrificed 72 hours following thefinal administration to determine the liver SRB-1 mRNA levels usingreal-time PCR and RIBOGREEN® RNA quantification reagent (MolecularProbes, Inc. Eugene, Oreg.) according to standard protocols. The resultsbelow are presented as the average percent of SRB-1 mRNA levels for eachtreatment group, normalized to the saline control.

As illustrated in Table 40, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner. Indeed, theantisense oligonucleotides comprising the phosphodiester linkedGalNAc₃-1 and GalNAc₃-9 conjugates at the 3′ terminus (ISIS 655861 andISIS 664078) and the GalNAc₃-3 and GalNAc₃-8 conjugates linked at the 5′terminus (ISIS 661161 and ISIS 665001) showed substantial improvement inpotency compared to the unconjugated antisense oligonucleotide (ISIS353382). Furthermore, ISIS 664078, comprising a GalNAc₃-9 conjugate atthe 3′ terminus was essentially equipotent compared to ISIS 655861,which comprises a GalNAc₃-1 conjugate at the 3′ terminus. The 5′conjugated antisense oligonucleotides, ISIS 661161 and ISIS 665001,comprising a GalNAc₃-3 or GalNAc₃-9, respectively, had increased potencycompared to the 3′ conjugated antisense oligonucleotides (ISIS 655861and ISIS 664078).

TABLE 40 ASOs containing GalNAc₃-1, 3, 8 or 9 targeting SRB-1 DosageSRB-1 mRNA ISIS No. (mg/kg) (% Saline) Conjugate Saline n/a 100 353382 388 none 10 68 30 36 655861 0.5 98 GalNac₃-1 (3′) 1.5 76 5 31 15 20664078 0.5 88 GalNac₃-9 (3′) 1.5 85 5 46 15 20 661161 0.5 92 GalNac₃-3(5′) 1.5 59 5 19 15 11 665001 0.5 100 GalNac₃-8 (5′) 1.5 73 5 29 15 13

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were measured relative to salineinjected mice using standard protocols. Total bilirubin and BUN werealso evaluated. The change in body weights was evaluated with nosignificant change from the saline group. ALTs, ASTs, total bilirubinand BUN values are shown in the table below.

TABLE 41 Dosage Total ISIS No. mg/kg ALT AST Bilirubin BUN ConjugateSaline 24 59 0.1 37.52 353382 3 21 66 0.2 34.65 none 10 22 54 0.2 34.230 22 49 0.2 33.72 655861 0.5 25 62 0.2 30.65 GalNac₃-1 (3′) 1.5 23 480.2 30.97 5 28 49 0.1 32.92 15 40 97 0.1 31.62 664078 0.5 40 74 0.1 35.3GalNac₃-9 (3′) 1.5 47 104 0.1 32.75 5 20 43 0.1 30.62 15 38 92 0.1 26.2661161 0.5 101 162 0.1 34.17 GalNac₃-3 (5′) 1.5 g 42 100 0.1 33.37   5 g23 99 0.1 34.97 15 53 83 0.1 34.8 665001 0.5 28 54 0.1 31.32 GalNac₃-8(5′) 1.5 42 75 0.1 32.32 5 24 42 0.1 31.85 15 32 67 0.1 31.

Example 56: Dose-Dependent Study of Oligonucleotides Comprising Either a3′ or 5′-Conjugate Group (Comparison of GalNAc₃-1, 2, 3, 5, 6, 7 and 10)Targeting SRB-1 In Vivo

The oligonucleotides listed below were tested in a dose-dependent studyfor antisense inhibition of SRB-1 in mice. Unconjugated ISIS 353382 wasincluded as a standard. Each of the various GalNAc₃ conjugate groups wasattached at the 5′ terminus of the respective oligonucleotide by aphosphodiester linked 2′-deoxyadenosine nucleoside (cleavable moiety)except for ISIS 655861 which had the GalNAc₃ conjugate group attached atthe 3′ terminus.

TABLE 42 Modified ASO targeting SRB-1 SEQ ASO Sequence (5′ to 3′) MotifConjugate ID No. ISIS 353382 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5no conjugate 4886 (parent) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) ISIS 655861 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5GalNAc ₃ -1 4887 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(eo) A _(do)′-GalNAc ₃ -1a ISIS 664507 GalNAc ₃ -2_(a) - _(o)′A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -2 4888^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) ISIS 661161 GalNAc ₃ -3 _(a) - _(o)′A _(do) 5/10/5GalNAc ₃ -3 4888 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 666224GalNAc ₃ -5 _(a) - _(o)′A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -5 4888^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) ISIS 666961 GalNAc ₃ -6 _(a) - _(o)′A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -64888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es)^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 666981 GalNAc ₃ -7 _(a) - _(o)′A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5GalNAc ₃ -7 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 666881GalNAc ₃ -10 _(a) - _(o)′A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -10 4888^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) Capital letters indicate the nucleobase for eachnucleoside and ^(m)C indicates a 5-methyl cytosine. Subscripts:“e” indicates a 2′-MOE modified nucleoside; “d” indicates aβ-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioateinternucleoside linkage (PS); “o” indicates a phosphodiesterinternucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—.Conjugate groups are in bold.

The structure of GalNAc₃-1_(a) was shown previously in Example 9. Thestructure of GalNAc₃-2_(a) was shown previously in Example 37. Thestructure of GalNAc₃-3_(a) was shown previously in Example 39. Thestructure of GalNAc₃-5_(a) was shown previously in Example 49. Thestructure of GalNAc₃-6_(a) was shown previously in Example 51. Thestructure of GalNAc₃-7_(a) was shown previously in Example 48. Thestructure of GalNAc₃-10_(a) was shown previously in Example 46.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected subcutaneously once at the dosage shown below with ISIS 353382,655861, 664507, 661161, 666224, 666961, 666981, 666881 or with saline.Each treatment group consisted of 4 animals. The mice were sacrificed 72hours following the final administration to determine the liver SRB-1mRNA levels using real-time PCR and RIBOGREEN RNA quantification reagent(Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols.The results below are presented as the average percent of SRB-1 mRNAlevels for each treatment group, normalized to the saline control.

As illustrated in Table 43, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner. Indeed, theconjugated antisense oligonucleotides showed substantial improvement inpotency compared to the unconjugated antisense oligonucleotide (ISIS353382). The 5′ conjugated antisense oligonucleotides showed a slightincrease in potency compared to the 3′ conjugated antisenseoligonucleotide.

TABLE 43 Dosage SRB-1 mRNA ISIS No. (mg/kg) (% Saline) Conjugate Salinen/a 100.0 353382 3 96.0 none 10 73.1 30 36.1 655861 0.5 99.4 GalNac₃-1(3′) 1.5 81.2 5 33.9 15 15.2 664507 0.5 102.0 GalNac₃-2 (5′) 1.5 73.2 531.3 15 10.8 661161 0.5 90.7 GalNac₃-3 (5′) 1.5 67.6 5 24.3 15 11.5666224 0.5 96.1 GalNac₃-5 (5′) 1.5 61.6 5 25.6 15 11.7 666961 0.5 85.5GalNAc₃-6 (5′) 1.5 56.3 5 34.2 15 13.1 666981 0.5 84.7 GalNAc₃-7 (5′)1.5 59.9 5 24.9 15 8.5 666881 0.5 100.0 GalNAc₃-10 (5′) 1.5 65.8 5 26.015 13.0

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were measured relative to salineinjected mice using standard protocols. Total bilirubin and BUN werealso evaluated. The change in body weights was evaluated with nosignificant change from the saline group. ALTs, ASTs, total bilirubinand BUN values are shown in Table 44 below.

TABLE 44 Dosage Total ISIS No. mg/kg ALT AST Bilirubin BUN ConjugateSaline 26 57 0.2 27 353382 3 25 92 0.2 27 none 10 23 40 0.2 25 30 29 540.1 28 655861 0.5 25 71 0.2 34 GalNac₃-1 (3′) 1.5 28 60 0.2 26 5 26 630.2 28 15 25 61 0.2 28 664507 0.5 25 62 0.2 25 GalNac₃-2 (5′) 1.5 24 490.2 26 5 21 50 0.2 26 15 59 84 0.1 22 661161 0.5 20 42 0.2 29 GalNac₃-3(5′) 1.5 g 37 74 0.2 25   5 g 28 61 0.2 29 15 21 41 0.2 25 666224 0.5 3448 0.2 21 GalNac₃-5 (5′) 1.5 23 46 0.2 26 5 24 47 0.2 23 15 32 49 0.1 26666961 0.5 17 63 0.2 26 GalNAc₃-6 (5′) 1.5 23 68 0.2 26 5 25 66 0.2 2615 29 107 0.2 28 666981 0.5 24 48 0.2 26 GalNAc₃-7 (5′) 1.5 30 55 0.2 245 46 74 0.1 24 15 29 58 0.1 26 666881 0.5 20 65 0.2 27 GalNAc₃-10 (5′)1.5 23 59 0.2 24 5 45 70 0.2 26 15 21 57 0.2 24

Example 57: Duration of Action Study of Oligonucleotides Comprising a3′-Conjugate Group Targeting ApoC III In Vivo

Mice were injected once with the doses indicated below and monitoredover the course of 42 days for ApoC-III and plasma triglycerides (PlasmaTG) levels. The study was performed using 3 transgenic mice that expresshuman APOC-III in each group.

TABLE 45 Modified ASO targeting ApoC III SEQ ID ASO Sequence (5′ to 3′)Linkages No. ISIS A_(es)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) PS 4878 304801 ^(m)C_(ds)^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) ISISA_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds)^(m)C_(ds) ^(m)C_(ds) PS 4879 647535 A_(ds)G_(ds)^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(eo) A _(do)′-GalNAc ₃ -1 _(a) ISISA_(es)G_(eo) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds)^(m)C_(ds) ^(m)C_(ds) PO/PS 4879 647536 A_(ds)G_(ds)^(m)C_(ds)T_(eo)T_(eo)T_(es)A_(es)T_(eo) A _(do)′-GalNAc ₃ -1 _(a)Capital letters indicate the nucleobase for each nucleoside and ^(m)Cindicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOEmodified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside;“s” indicates a phosphorothioate internucleoside linkage (PS);“o” indicates a phosphodiester internucleoside linkage (PO); and“o′” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-1_(a) was shown previously in Example 9.

TABLE 46 ApoC III mRNA (% Saline on Day 1) and Plasma TG Levels (%Saline on Day 1) ASO Dose Target Day 3 Day 7 Day 14 Day 35 Day 42 Saline 0 mg/kg ApoC-III 98 100 100 95 116 ISIS 30 mg/kg ApoC-III 28 30 41 6574 304801 ISIS 10 mg/kg ApoC-III 16 19 25 74 94 647535 ISIS 10 mg/kgApoC-III 18 16 17 35 51 647536 Saline  0 mg/kg Plasma TG 121 130 123 105109 ISIS 30 mg/kg Plasma TG 34 37 50 69 69 304801 ISIS 10 mg/kg PlasmaTG 18 14 24 18 71 647535 ISIS 10 mg/kg Plasma TG 21 19 15 32 35 647536

As can be seen in the table above the duration of action increased withaddition of the 3′-conjugate group compared to the unconjugatedoligonucleotide. There was a further increase in the duration of actionfor the conjugated mixed PO/PS oligonucleotide 647536 as compared to theconjugated full PS oligonucleotide 647535.

Example 58: Dose-Dependent Study of Oligonucleotides Comprising a3′-Conjugate Group (Comparison of GalNAc₃-1 and GalNAc₄-11) TargetingSRB-1 In Vivo

The oligonucleotides listed below were tested in a dose-dependent studyfor antisense inhibition of SRB-1 in mice. Unconjugated ISIS 440762 wasincluded as an unconjugated standard. Each of the conjugate groups wereattached at the 3′ terminus of the respective oligonucleotide by aphosphodiester linked 2′-deoxyadenosine nucleoside cleavable moiety.

The structure of GalNAc₃-1_(a) was shown previously in Example 9. Thestructure of GalNAc₃-11_(a) was shown previously in Example 50.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected subcutaneously once at the dosage shown below with ISIS 440762,651900, 663748 or with saline. Each treatment group consisted of 4animals. The mice were sacrificed 72 hours following the finaladministration to determine the liver SRB-1 mRNA levels using real-timePCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc.Eugene, Oreg.) according to standard protocols. The results below arepresented as the average percent of SRB-1 mRNA levels for each treatmentgroup, normalized to the saline control.

As illustrated in Table 47, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner. The antisenseoligonucleotides comprising the phosphodiester linked GalNAc₃-1 andGalNAc₄-11 conjugates at the 3′ terminus (ISIS 651900 and ISIS 663748)showed substantial improvement in potency compared to the unconjugatedantisense oligonucleotide (ISIS 440762). The two conjugatedoligonucleotides, GalNAc₃-1 and GalNAc₄-11, were equipotent.

TABLE 47 Modified ASO targeting SRB-1 % Saline SEQ ID ASO Sequence(5′ to 3′) Dose mg/kg control No. Saline 100 ISIS 440762 T_(ks)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 0.673.45 4880 ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) 2 59.66 6 23.50 ISIS 651900T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)0.2 62.75 4881 ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(ko) A _(do)′-GalNAc ₃ -1_(a) 0.6 29.14 2 8.61 6 5.62 ISIS 663748 T_(ks)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 0.263.99 4881 ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(ko) A _(do)′-GalNAc ₄ -11 _(a)0.6 33.53 2 7.58 6 5.52 Capital letters indicate the nucleobase for eachnucleoside and ^(m)C indicates a 5-methyl cytosine. Subscripts:“e” indicates a 2′-MOE modified nucleoside; “k” indicates 6′-(S)—CH₃bicyclic nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside;“s” indicates a phosphorothioate internucleoside linkage (PS);“o” indicates a phosphodiester internucleoside linkage (PO); and“o′” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were measured relative to salineinjected mice using standard protocols. Total bilirubin and BUN werealso evaluated. The change in body weights was evaluated with nosignificant change from the saline group. ALTs, ASTs, total bilirubinand BUN values are shown in Table 48 below.

TABLE 48 Dosage Total ISIS No. mg/kg ALT AST Bilirubin BUN ConjugateSaline 30 76 0.2 40 440762 0.60 32 70 0.1 35 none 2 26 57 0.1 35 6 31 480.1 39 651900 0.2 32 115 0.2 39 GalNac₃-1 (3′) 0.6 33 61 0.1 35 2 30 500.1 37 6 34 52 0.1 36 663748 0.2 28 56 0.2 36 GalNac₄-11 (3′) 0.6 34 600.1 35 2 44 62 0.1 36 6 38 71 0.1 33

Example 59: Effects of GalNAc₃-1 Conjugated ASOs Targeting FXI In Vivo

The oligonucleotides listed below were tested in a multiple dose studyfor antisense inhibition of FXI in mice. ISIS 404071 was included as anunconjugated standard. Each of the conjugate groups was attached at the3′ terminus of the respective oligonucleotide by a phosphodiester linked2′-deoxyadenosine nucleoside cleavable moiety.

TABLE 49 Modified ASOs targeting FXI SEQ ID ASO Sequence (5′ to 3′)Linkages No. ISIS T_(es)G_(es)G_(es)T_(es)A_(es)A_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds) ^(m)C_(ds) PS 4889 404071 T_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(es)G_(es)A_(es)G_(es)G_(e) ISIST_(es)G_(es)G_(es)T_(es)A_(es)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)^(m)C_(ds) PS 4890 656172 T_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(es)G_(es)A_(es)G_(es)G_(eo) A _(do)′-GalNAc ₃ -1 _(a) ISIST_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)^(m)C_(ds) PO/PS 4890 656173 T_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(eo) A _(do)′-GalNAc ₃ -1 _(a)Capital letters indicate the nucleobase for each nucleoside and ^(m)Cindicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOEmodified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside;“s” indicates a phosphorothioate internucleoside linkage (PS);“o” indicates a phosphodiester internucleoside linkage (PO); and“o′” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-1_(a) was shown previously in Example 9.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected subcutaneously twice a week for 3 weeks at the dosage shownbelow with ISIS 404071, 656172, 656173 or with PBS treated control. Eachtreatment group consisted of 4 animals. The mice were sacrificed 72hours following the final administration to determine the liver FXI mRNAlevels using real-time PCR and RIBOGREEN RNA quantification reagent(Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols.Plasma FXI protein levels were also measured using ELISA. FXI mRNAlevels were determined relative to total RNA (using RIBOGREEN®), priorto normalization to PBS-treated control. The results below are presentedas the average percent of FXI mRNA levels for each treatment group. Thedata was normalized to PBS-treated control and is denoted as “% PBS”.The ED₅₀s were measured using similar methods as described previouslyand are presented below.

TABLE 50 Factor XI mRNA (% Saline) Dose ASO mg/kg % Control ConjugateLinkages Saline 100 none ISIS 3 92 none PS 404071 10 40 30 15 ISIS 0.774 GalNAc₃-1 PS 656172 2 33 6 9 ISIS 0.7 49 GalNAc₃-1 PO/PS 656173 2 226 1

As illustrated in Table 50, treatment with antisense oligonucleotideslowered FXI mRNA levels in a dose-dependent manner. The oligonucleotidescomprising a 3′-GalNAc₃-1 conjugate group showed substantial improvementin potency compared to the unconjugated antisense oligonucleotide (ISIS404071). Between the two conjugated oligonucleotides an improvement inpotency was further provided by substituting some of the PS linkageswith PO (ISIS 656173).

As illustrated in Table 50a, treatment with antisense oligonucleotideslowered FXI protein levels in a dose-dependent manner. Theoligonucleotides comprising a 3′-GalNAc₃-1 conjugate group showedsubstantial improvement in potency compared to the unconjugatedantisense oligonucleotide (ISIS 404071). Between the two conjugatedoligonucleotides an improvement in potency was further provided bysubstituting some of the PS linkages with PO (ISIS 656173).

TABLE 50a Factor XI protein (% Saline) Dose Protein ASO mg/kg (%Control) Conjugate Linkages Saline 100 none ISIS 3 127 none PS 404071 1032 30 3 ISIS 0.7 70 GalNAc₃-1 PS 656172 2 23 6 1 ISIS 0.7 45 GalNAc₃-1PO/PS 656173 2 6 6 0

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were measured relative to salineinjected mice using standard protocols. Total bilirubin, total albumin,CRE and BUN were also evaluated. The change in body weights wasevaluated with no significant change from the saline group. ALTs, ASTs,total bilirubin and BUN values are shown in the table below.

TABLE 51 Dosage Total Total ISIS No. mg/kg ALT AST Albumin Bilirubin CREBUN Conjugate Saline 71.8 84.0 3.1 0.2 0.2 22.9 404071 3 152.8 176.0 3.10.3 0.2 23.0 none 10 73.3 121.5 3.0 0.2 0.2 21.4 30 82.5 92.3 3.0 0.20.2 23.0 656172 0.7 62.5 111.5 3.1 0.2 0.2 23.8 GalNac₃-1 (3′) 2 33.051.8 2.9 0.2 0.2 22.0 6 65.0 71.5 3.2 0.2 0.2 23.9 656173 0.7 54.8 90.53.0 0.2 0.2 24.9 GalNac₃-1 (3′) 2 85.8 71.5 3.2 0.2 0.2 21.0 6 114.0101.8 3.3 0.2 0.2 22.7

Example 60: Effects of Conjugated ASOs Targeting SRB-1 In Vitro

The oligonucleotides listed below were tested in a multiple dose studyfor antisense inhibition of SRB-1 in primary mouse hepatocytes. ISIS353382 was included as an unconjugated standard. Each of the conjugategroups were attached at the 3′ or 5′ terminus of the respectiveoligonucleotide by a phosphodiester linked 2′-deoxyadenosine nucleosidecleavable moiety.

TABLE 52 Modified ASO targeting SRB-1 SEQ ASO Sequence (5′ to 3′) MotifConjugate ID No. ISIS 353382 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5none 4886 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS655861 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 GalNAc ₃ -1 4887^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do)′-GalNAc₃ -1 _(a) ISIS 655862 G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5GalNAc ₃ -1 4887 ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo)^(m)C_(es)T_(es)T_(eo) A _(do)′-GalNAc ₃ -1 _(a) ISIS 661161 GalNAc ₃ -3_(a-o)′A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)5/10/5 GalNAc ₃ -3 4888 T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 665001GalNAc ₃ -8 _(a-o)′A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds) 5/10/5 GalNAc ₃ -8 4888 T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) ISIS 664078 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5GalNAc ₃ -9 4887 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(eo) A _(do)′-GalNAc ₃ -9 _(a) ISIS 666961 GalNAC ₃ -6_(a) - _(o)′A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)5/10/5 GalNAc ₃ -6 4888 T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 664507GalNAc ₃ -2 _(a) - _(o)′A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -2 4888^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) ISIS 666881 GalNAc ₃ -10 _(a) - _(o)′A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -104888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es)^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 666224 GalNAc ₃ -5 _(a) - _(o)′A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5GalNAc ₃ -5 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 666981GalNAc ₃ -7 _(a) - _(o)′A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -7 4888^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) Capital letters indicate the nucleobase for eachnucleoside and ^(m)C indicates a 5-methyl cytosine. Subscripts:“e” indicates a 2′-MOE modified nucleoside; “d” indicates aβ-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioateinternucleoside linkage (PS); “o” indicates a phosphodiesterinternucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—.Conjugate groups are in bold.

The structure of GalNAc₃-1_(a) was shown previously in Example 9. Thestructure of GalNAc₃-3a was shown previously in Example 39. Thestructure of GalNAc₃-8a was shown previously in Example 47. Thestructure of GalNAc₃-9a was shown previously in Example 52. Thestructure of GalNAc₃-6a was shown previously in Example 51. Thestructure of GalNAc₃-2a was shown previously in Example 37. Thestructure of GalNAc₃-10a was shown previously in Example 46. Thestructure of GalNAc₃-5a was shown previously in Example 49. Thestructure of GalNAc₃-7a was shown previously in Example 48.

Treatment

The oligonucleotides listed above were tested in vitro in primary mousehepatocyte cells plated at a density of 25,000 cells per well andtreated with 0.03, 0.08, 0.24, 0.74, 2.22, 6.67 or 20 nM modifiedoligonucleotide. After a treatment period of approximately 16 hours, RNAwas isolated from the cells and mRNA levels were measured byquantitative real-time PCR and the SRB-1 mRNA levels were adjustedaccording to total RNA content, as measured by RIBOGREEN®.

The IC₅₀ was calculated using standard methods and the results arepresented in Table 53. The results show that, under free uptakeconditions in which no reagents or electroporation techniques are usedto artificially promote entry of the oligonucleotides into cells, theoligonucleotides comprising a GalNAc conjugate were significantly morepotent in hepatocytes than the parent oligonucleotide (ISIS 353382) thatdoes not comprise a GalNAc conjugate.

TABLE 53 IC₅₀ Internucleoside SEQ ASO (nM) linkages Conjugate ID No.ISIS 353382   190^(a) PS none 4886 ISIS 655861  11^(a) PS GalNAc₃-1 4887ISIS 655862  3 PO/PS GalNAc₃-1 4887 ISIS 661161  15^(a) PS GalNAc₃-34888 ISIS 665001 20 PS GalNAc₃-8 4888 ISIS 664078 55 PS GalNAc₃-9 4887ISIS 666961  22^(a) PS GalNAc₃-6 4888 ISIS 664507 30 PS GalNAc₃-2 4888ISIS 666881 30 PS GalNAc₃-10 4888 ISIS 666224  30^(a) PS GalNAc₃-5 4888ISIS 666981 40 PS GalNAc₃-7 4888 ^(a)Average of multiple runs.

Example 61: Preparation of Oligomeric Compound 175 Comprising GalNAc₃-12

Compound 169 is commercially available. Compound 172 was prepared byaddition of benzyl (perfluorophenyl) glutarate to compound 171. Thebenzyl (perfluorophenyl) glutarate was prepared by adding PFP-TFA andDIEA to 5-(benzyloxy)-5-oxopentanoic acid in DMF. Oligomeric compound175, comprising a GalNAc₃-12 conjugate group, was prepared from compound174 using the general procedures illustrated in Example 46. The GalNAc₃cluster portion of the conjugate group GalNAc₃-12 (GalNAc₃-12_(a)) canbe combined with any cleavable moiety to provide a variety of conjugategroups. In a certain embodiments, the cleavable moiety is—P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-12(GalNAc₃-12_(a)-CM-) is shown below:

Example 62: Preparation of Oligomeric Compound 180 Comprising GalNAc₃-13

Compound 176 was prepared using the general procedure shown in Example2. Oligomeric compound 180, comprising a GalNAc₃-13 conjugate group, wasprepared from compound 177 using the general procedures illustrated inExample 49. The GalNAc₃ cluster portion of the conjugate groupGalNAc₃-13 (GalNAc₃-13a) can be combined with any cleavable moiety toprovide a variety of conjugate groups. In a certain embodiments, thecleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure ofGalNAc₃-13 (GalNAc₃-13_(a)-CM-) is shown below:

Example 63: Preparation of Oligomeric Compound 188 Comprising GalNAc₃-14

Compounds 181 and 185 are commercially available. Oligomeric compound188, comprising a GalNAc₃-14 conjugate group, was prepared from compound187 using the general procedures illustrated in Example 46. The GalNAc₃cluster portion of the conjugate group GalNAc₃-14 (GalNAc₃-14_(a)) canbe combined with any cleavable moiety to provide a variety of conjugategroups. In certain embodiments, the cleavable moiety is—P(O)(OH)-A_(d)-P(O)(OH)—. The structure of GalNAc₃-14(GalNAc₃-14_(a)-CM-) is shown below:

Example 64: Preparation of Oligomeric Compound 197 Comprising GalNAc₃-15

Compound 189 is commercially available. Compound 195 was prepared usingthe general procedure shown in Example 31. Oligomeric compound 197,comprising a GalNAc₃-15 conjugate group, was prepared from compounds 194and 195 using standard oligonucleotide synthesis procedures. The GalNAc₃cluster portion of the conjugate group GalNAc₃-15 (GalNAc₃-15_(a)) canbe combined with any cleavable moiety to provide a variety of conjugategroups. In certain embodiments, the cleavable moiety is—P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-15(GalNAc₃-15_(a)-CM-) is shown below:

Example 65: Dose-Dependent Study of Oligonucleotides Comprising a5′-Conjugate Group (Comparison of GalNAc₃-3, 12, 13, 14, and 15)Targeting SRB-1 In Vivo

The oligonucleotides listed below were tested in a dose-dependent studyfor antisense inhibition of SRB-1 in mice. Unconjugated ISIS 353382 wasincluded as a standard. Each of the GalNAc₃ conjugate groups wasattached at the 5′ terminus of the respective oligonucleotide by aphosphodiester linked 2′-deoxyadenosine nucleoside (cleavable moiety).

TABLE 54 Modified ASOs targeting SRB-1 SEQ ISIS ID No. Sequences (5′ to3′) Conjugate No. 353382 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) none 4886 661161GalNAc ₃ -3 _(a) - _(o)′A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds) GalNAc₃-3 4888 T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)671144 GalNAc ₃ -12 _(a) - _(o)′A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds) GalNAc₃-12 4888 T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)670061 GalNAc ₃ -13 _(a) - _(o)′A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds) GalNAc₃-13 4888 T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)671261 GalNAc ₃ -14 _(a) - _(o)′A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds) GalNAc₃-14 4888 T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)671262 GalNAc₃-15_(a)-_(o)′A_(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds) GalNAc₃-15 4888 T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)Capital letters indicate the nucleobase for each nucleoside and ^(m)Cindicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOEmodified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside;“s” indicates a phosphorothioate internucleoside linkage (PS);“o” indicates a phosphodiester internucleoside linkage (PO); and“o′” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-3_(a) was shown previously in Example 39. Thestructure of GalNAc₃-12a was shown previously in Example 61. Thestructure of GalNAc₃-13a was shown previously in Example 62. Thestructure of GalNAc₃-14a was shown previously in Example 63. Thestructure of GalNAc₃-15a was shown previously in Example 64.

Treatment

Six to eight week old C57bl6 mice (Jackson Laboratory, Bar Harbor, Me.)were injected subcutaneously once or twice at the dosage shown belowwith ISIS 353382, 661161, 671144, 670061, 671261, 671262, or withsaline. Mice that were dosed twice received the second dose three daysafter the first dose. Each treatment group consisted of 4 animals. Themice were sacrificed 72 hours following the final administration todetermine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN®RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.)according to standard protocols. The results below are presented as theaverage percent of SRB-1 mRNA levels for each treatment group,normalized to the saline control.

As illustrated in Table 55, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner. No significantdifferences in target knockdown were observed between animals thatreceived a single dose and animals that received two doses (see ISIS353382 dosages 30 and 2×15 mg/kg; and ISIS 661161 dosages 5 and 2×2.5mg/kg). The antisense oligonucleotides comprising the phosphodiesterlinked GalNAc₃-3, 12, 13, 14, and 15 conjugates showed substantialimprovement in potency compared to the unconjugated antisenseoligonucleotide (ISIS 335382).

TABLE 55 SRB-1 mRNA (% Saline) Dosage SRB-1 mRNA ED₅₀ ISIS No. (mg/kg)(% Saline) (mg/kg) Conjugate Saline n/a 100.0 n/a n/a 353382 3 85.0 22.4none 10 69.2 30 34.2 2 × 15  36.0 661161 0.5 87.4 2.2 GalNAc₃-3 1.5 59.05 25.6 2 × 2.5 27.5 15 17.4 671144 0.5 101.2 3.4 GalNAc₃-12 1.5 76.1 532.0 15 17.6 670061 0.5 94.8 2.1 GalNAc₃-13 1.5 57.8 5 20.7 15 13.3671261 0.5 110.7 4.1 GalNAc₃-14 1.5 81.9 5 39.8 15 14.1 671262 0.5 109.49.8 GalNAc₃-15 1.5 99.5 5 69.2 15 36.1

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were measured relative to salineinjected mice using standard protocols. Total bilirubin and BUN werealso evaluated. The changes in body weights were evaluated with nosignificant differences from the saline group (data not shown). ALTs,ASTs, total bilirubin and BUN values are shown in Table 56 below.

TABLE 56 Total Dosage ALT AST Bilirubin BUN ISIS No. (mg/kg) (U/L) (U/L)(mg/dL) (mg/dL) Conjugate Saline n/a 28 60 0.1 39 n/a 353382 3 30 77 0.236 none 10 25 78 0.2 36 30 28 62 0.2 35 2 × 15  22 59 0.2 33 661161 0.539 72 0.2 34 GalNAc₃-3 1.5 26 50 0.2 33 5 41 80 0.2 32 2 × 2.5 24 72 0.228 15 32 69 0.2 36 671144 0.5 25 39 0.2 34 GalNAc₃-12 1.5 26 55 0.2 28 548 82 0.2 34 15 23 46 0.2 32 670061 0.5 27 53 0.2 33 GalNAc₃-13 1.5 2445 0.2 35 5 23 58 0.1 34 15 24 72 0.1 31 671261 0.5 69 99 0.1 33GalNAc₃-14 1.5 34 62 0.1 33 5 43 73 0.1 32 15 32 53 0.2 30 671262 0.5 2451 0.2 29 GalNAc₃-15 1.5 32 62 0.1 31 5 30 76 0.2 32 15 31 64 0.1 32

Example 66: Effect of Various Cleavable Moieties on Antisense InhibitionIn Vivo by Oligonucleotides Targeting SRB-1 Comprising a 5′-GalNAc₃Cluster

The oligonucleotides listed below were tested in a dose-dependent studyfor antisense inhibition of SRB-1 in mice. Each of the GalNAc₃ conjugategroups was attached at the 5′ terminus of the respective oligonucleotideby a phosphodiester linked nucleoside (cleavable moiety (CM)).

TABLE 57 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 661161 GalNAc ₃ -3 _(a) - _(o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a A_(d) 4888 G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 670699 GalNAc ₃-3 _(a) - _(o′) T _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a T_(d)4891 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo)C_(es)T_(es)T_(e)670700 GalNAc ₃ -3 _(a) - _(o′) A _(eo)G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a A_(e)4888 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo)^(m)C_(es)T_(es)T_(e) 670701 GalNAC ₃ -3 _(a) - _(o′) T _(eo)G_(es)^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a T_(e) 4891 G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 671165 GalNAc ₃-13 _(a) - _(o′) A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-13a A_(d)4888 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo)^(m)C_(es)T_(es)T_(e) Capital letters indicate the nucleobase for eachnucleoside and ^(m)C indicates a 5-methyl cytosine. Subscripts:“e” indicates a 2′-MOE modified nucleoside; “d” indicates aβ-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioateinternucleoside linkage (PS); “o” indicates a phosphodiesterinternucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—.Conjugate groups are in bold.

The structure of GalNAc₃-3_(a) was shown previously in Example 39. Thestructure of GalNAc₃-13a was shown previously in Example 62.

Treatment

Six to eight week old C57bl6 mice (Jackson Laboratory, Bar Harbor, Me.)were injected subcutaneously once at the dosage shown below with ISIS661161, 670699, 670700, 670701, 671165, or with saline. Each treatmentgroup consisted of 4 animals. The mice were sacrificed 72 hoursfollowing the final administration to determine the liver SRB-1 mRNAlevels using real-time PCR and RIBOGREEN® RNA quantification reagent(Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols.The results below are presented as the average percent of SRB-1 mRNAlevels for each treatment group, normalized to the saline control.

As illustrated in Table 58, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner. The antisenseoligonucleotides comprising various cleavable moieties all showedsimilar potencies.

TABLE 58 SRB-1 mRNA (% Saline) Dosage SRB-1 mRNA GalNAc₃ ISIS No.(mg/kg) (% Saline) Cluster CM Saline n/a 100.0 n/a n/a 661161 0.5 87.8GalNAc₃-3a A_(d) 1.5 61.3 5 33.8 15 14.0 670699 0.5 89.4 GalNAc₃-3aT_(d) 1.5 59.4 5 31.3 15 17.1 670700 0.5 79.0 GalNAc₃-3a A_(e) 1.5 63.35 32.8 15 17.9 670701 0.5 79.1 GalNAc₃-3a T_(e) 1.5 59.2 5 35.8 15 17.7671165 0.5 76.4 GalNAc₃-13a A_(d) 1.5 43.2 5 22.6 15 10.0

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were measured relative to salineinjected mice using standard protocols. Total bilirubin and BUN werealso evaluated. The changes in body weights were evaluated with nosignificant differences from the saline group (data not shown). ALTs,ASTs, total bilirubin and BUN values are shown in Table 56 below.

TABLE 59 Total Dosage ALT AST Bilirubin BUN GalNAc₃ ISIS No. (mg/kg)(U/L) (U/L) (mg/dL) (mg/dL) Cluster CM Saline n/a 24 64 0.2 31 n/a n/a661161 0.5 25 64 0.2 31 GalNAc₃-3a A_(d) 1.5 24 50 0.2 32 5 26 55 0.2 2815 27 52 0.2 31 670699 0.5 42 83 0.2 31 GalNAc₃-3a T_(d) 1.5 33 58 0.232 5 26 70 0.2 29 15 25 67 0.2 29 670700 0.5 40 74 0.2 27 GalNAc₃-3aA_(e) 1.5 23 62 0.2 27 5 24 49 0.2 29 15 25 87 0.1 25 670701 0.5 30 770.2 27 GalNAc₃-3a T_(e) 1.5 22 55 0.2 30 5 81 101 0.2 25 15 31 82 0.2 24671165 0.5 44 84 0.2 26 GalNAc₃-13a A_(d) 1.5 47 71 0.1 24 5 33 91 0.226 15 33 56 0.2 29

Example 67: Preparation of Oligomeric Compound 199 Comprising GalNAc₃-16

Oligomeric compound 199, comprising a GalNAc₃-16 conjugate group, isprepared using the general procedures illustrated in Examples 7 and 9.The GalNAc₃ cluster portion of the conjugate group GalNAc₃-16(GalNAc₃-16_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. In certain embodiments, the cleavablemoiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-16(GalNAc₃-16_(a)-CM-) is shown below:

Example 68: Preparation of Oligomeric Compound 200 Comprising GalNAc₃-17

Oligomeric compound 200, comprising a GalNAc₃-17 conjugate group, wasprepared using the general procedures illustrated in Example 46. TheGalNAc₃ cluster portion of the conjugate group GalNAc₃-17(GalNAc₃-17_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. In certain embodiments, the cleavablemoiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-17(GalNAc₃-17_(a)-CM-) is shown below:

Example 69: Preparation of Oligomeric Compound 201 Comprising GalNAc₃-18

Oligomeric compound 201, comprising a GalNAc₃-18 conjugate group, wasprepared using the general procedures illustrated in Example 46. TheGalNAc₃ cluster portion of the conjugate group GalNAc₃-18(GalNAc₃-18_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. In certain embodiments, the cleavablemoiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-18(GalNAc₃-18_(a)-CM-) is shown below:

Example 70: Preparation of Oligomeric Compound 204 Comprising GalNAc₃-19

Oligomeric compound 204, comprising a GalNAc₃-19 conjugate group, wasprepared from compound 64 using the general procedures illustrated inExample 52. The GalNAc₃ cluster portion of the conjugate groupGalNAc₃-19 (GalNAc₃-19_(a)) can be combined with any cleavable moiety toprovide a variety of conjugate groups. In certain embodiments, thecleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure ofGalNAc₃-19 (GalNAc₃-19_(a)-CM-) is shown below:

Example 71: Preparation of Oligomeric Compound 210 Comprising GalNAc₃-20

Compound 205 was prepared by adding PFP-TFA and DIEA to6-(2,2,2-trifluoroacetamido)hexanoic acid in acetonitrile, which wasprepared by adding triflic anhydride to 6-aminohexanoic acid. Thereaction mixture was heated to 80° C., then lowered to rt. Oligomericcompound 210, comprising a GalNAc₃-20 conjugate group, was prepared fromcompound 208 using the general procedures illustrated in Example 52. TheGalNAc₃ cluster portion of the conjugate group GalNAc₃-20(GalNAc₃-20_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. In certain embodiments, the cleavablemoiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-20(GalNAc₃-20_(a)-CM-) is shown below:

Example 72: Preparation of Oligomeric Compound 215 Comprising GalNAc₃-21

Compound 211 is commercially available. Oligomeric compound 215,comprising a GalNAc₃-21 conjugate group, was prepared from compound 213using the general procedures illustrated in Example 52. The GalNAc₃cluster portion of the conjugate group GalNAc₃-21 (GalNAc₃-21_(a)) canbe combined with any cleavable moiety to provide a variety of conjugategroups. In certain embodiments, the cleavable moiety is—P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-21(GalNAc₃-2_(a)-CM-) is shown below:

Example 73: Preparation of Oligomeric Compound 221 Comprising GalNAc₃-22

Compound 220 was prepared from compound 219 using diisopropylammoniumtetrazolide. Oligomeric compound 221, comprising a GalNAc₃-21 conjugategroup, is prepared from compound 220 using the general procedureillustrated in Example 52. The GalNAc₃ cluster portion of the conjugategroup GalNAc₃-22 (GalNAc₃-22_(a)) can be combined with any cleavablemoiety to provide a variety of conjugate groups. In certain embodiments,the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure ofGalNAc₃-22 (GalNAc₃-22_(a)-CM-) is shown below:

Example 74: Effect of Various Cleavable Moieties on Antisense InhibitionIn Vivo by Oligonucleotides Targeting SRB-1 Comprising a 5′-GalNAc₃Conjugate

The oligonucleotides listed below were tested in a dose-dependent studyfor antisense inhibition of SRB-1 in mice. Each of the GalNAc₃ conjugategroups was attached at the 5′ terminus of the respectiveoligonucleotide.

TABLE 60 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 353382 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) n/a n/a 4886 ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)661161 GalNAc ₃ -3 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a A_(d)4888 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) 666904 GalNAc ₃ -3 _(a) - _(o′)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a PO 4886 G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 675441 GalNAc ₃-17 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-17a A_(d)4888 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) 675442 GalNAc ₃ -18 _(a) - _(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)C_(ds)A_(ds)T_(ds) GalNAc₃-18a A_(d) 4888 G_(ds)A_(ds)^(m)C_(ds)T_(ds) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)

In all tables, capital letters indicate the nucleobase for eachnucleoside and C indicates a 5-methyl cytosine. Subscripts: “e”indicates a 2′-MOE modified nucleoside; “d” indicates aβ-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioateinternucleoside linkage (PS); “o” indicates a phosphodiesterinternucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—.Conjugate groups are in bold.

The structure of GalNAc₃-3_(a) was shown previously in Example 39. Thestructure of GalNAc₃-17a was shown previously in Example 68, and thestructure of GalNAc₃-18a was shown in Example 69.

Treatment

Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.)were injected subcutaneously once at the dosage shown below with anoligonucleotide listed in Table 60 or with saline. Each treatment groupconsisted of 4 animals. The mice were sacrificed 72 hours following thefinal administration to determine the SRB-1 mRNA levels using real-timePCR and RIBOGREEN RNA quantification reagent (Molecular Probes, Inc.Eugene, Oreg.) according to standard protocols. The results below arepresented as the average percent of SRB-1 mRNA levels for each treatmentgroup, normalized to the saline control.

As illustrated in Table 61, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner. The antisenseoligonucleotides comprising a GalNAc conjugate showed similar potenciesand were significantly more potent than the parent oligonucleotidelacking a GalNAc conjugate.

TABLE 61 SRB-1 mRNA (% Saline) Dosage SRB-1 mRNA GalNAc₃ ISIS No.(mg/kg) (% Saline) Cluster CM Saline n/a 100.0 n/a n/a 353382 3 79.38n/a n/a 10 68.67 30 40.70 661161 0.5 79.18 GalNAc₃-3a A_(d) 1.5 75.96 530.53 15 12.52 666904 0.5 91.30 GalNAc₃-3a PO 1.5 57.88 5 21.22 15 16.49675441 0.5 76.71 GalNAc₃-17a A_(d) 1.5 63.63 5 29.57 15 13.49 675442 0.595.03 GalNAc₃-18a A_(d) 1.5 60.06 5 31.04 15 19.40

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were measured relative to salineinjected mice using standard protocols. Total bilirubin and BUN werealso evaluated. The change in body weights was evaluated with nosignificant change from the saline group (data not shown). ALTs, ASTs,total bilirubin and BUN values are shown in Table 62 below.

TABLE 62 Total Dosage ALT AST Bilirubin BUN GalNAc₃ ISIS No. (mg/kg)(U/L) (U/L) (mg/dL) (mg/dL) Cluster CM Saline n/a 26 59 0.16 42 n/a n/a353382 3 23 58 0.18 39 n/a n/a 10 28 58 0.16 43 30 20 48 0.12 34 6611610.5 30 47 0.13 35 GalNAc₃-3a A_(d) 1.5 23 53 0.14 37 5 26 48 0.15 39 1532 57 0.15 42 666904 0.5 24 73 0.13 36 GalNAc₃-3a PO 1.5 21 48 0.12 32 519 49 0.14 33 15 20 52 0.15 26 675441 0.5 42 148 0.21 36 GalNAc₃-17aA_(d) 1.5 60 95 0.16 34 5 27 75 0.14 37 15 24 61 0.14 36 675442 0.5 2665 0.15 37 GalNAc₃-18a A_(d) 1.5 25 64 0.15 43 5 27 69 0.15 37 15 30 840.14 37

Example 75: Pharmacokinetic Analysis of Oligonucleotides Comprising a5′-Conjugate Group

The PK of the ASOs in Tables 54, 57 and 60 above was evaluated usingliver samples that were obtained following the treatment proceduresdescribed in Examples 65, 66, and 74. The liver samples were minced andextracted using standard protocols and analyzed by IP-HPLC-MS alongsidean internal standard. The combined tissue level (μg/g) of allmetabolites was measured by integrating the appropriate UV peaks, andthe tissue level of the full-length ASO missing the conjugate (“parent,”which is Isis No. 353382 in this case) was measured using theappropriate extracted ion chromatograms (EIC).

TABLE 63 PK Analysis in Liver Parent ASO Total Tissue Tissue LevelDosage Level by UV by EIC GalNAc₃ ISIS No. (mg/kg) (μg/g) (μg/g) ClusterCM 353382 3 8.9 8.6 n/a n/a 10 22.4 21.0 30 54.2 44.2 661161 5 32.4 20.7GalNAc₃-3a A_(d) 15 63.2 44.1 671144 5 20.5 19.2 GalNAc₃-12a A_(d) 1548.6 41.5 670061 5 31.6 28.0 GalNAc₃-13a A_(d) 15 67.6 55.5 671261 519.8 16.8 GalNAc₃-14a A_(d) 15 64.7 49.1 671262 5 18.5 7.4 GalNAc₃-15aA_(d) 15 52.3 24.2 670699 5 16.4 10.4 GalNAc₃-3a T_(d) 15 31.5 22.5670700 5 19.3 10.9 GalNAc₃-3a A_(e) 15 38.1 20.0 670701 5 21.8 8.8GalNAc₃-3a T_(e) 15 35.2 16.1 671165 5 27.1 26.5 GalNAc₃-13a A_(d) 1548.3 44.3 666904 5 30.8 24.0 GalNAc₃-3a PO 15 52.6 37.6 675441 5 25.419.0 GalNAc₃-17a A_(d) 15 54.2 42.1 675442 5 22.2 20.7 GalNAc₃-18a A_(d)15 39.6 29.0

The results in Table 63 above show that there were greater liver tissuelevels of the oligonucleotides comprising a GalNAc₃ conjugate group thanof the parent oligonucleotide that does not comprise a GalNAc₃ conjugategroup (ISIS 353382) 72 hours following oligonucleotide administration,particularly when taking into consideration the differences in dosingbetween the oligonucleotides with and without a GalNAc₃ conjugate group.Furthermore, by 72 hours, 40-98% of each oligonucleotide comprising aGalNAc₃ conjugate group was metabolized to the parent compound,indicating that the GalNAc₃ conjugate groups were cleaved from theoligonucleotides.

Example 76: Preparation of Oligomeric Compound 230 Comprising GalNAc₃-23

Compound 222 is commercially available. 44.48 ml (0.33 mol) of compound222 was treated with tosyl chloride (25.39 g, 0.13 mol) in pyridine (500mL) for 16 hours. The reaction was then evaporated to an oil, dissolvedin EtOAc and washed with water, sat. NaHCO₃, brine, and dried overNa₂SO₄. The ethyl acetate was concentrated to dryness and purified bycolumn chromatography, eluted with EtOAc/hexanes (1:1) followed by 10%methanol in CH₂Cl₂ to give compound 223 as a colorless oil. LCMS and NMRwere consistent with the structure. 10 g (32.86 mmol) of1-Tosyltriethylene glycol (compound 223) was treated with sodium azide(10.68 g, 164.28 mmol) in DMSO (100 mL) at room temperature for 17hours. The reaction mixture was then poured onto water, and extractedwith EtOAc. The organic layer was washed with water three times anddried over Na₂SO₄. The organic layer was concentrated to dryness to give5.3 g of compound 224 (92%). LCMS and NMR were consistent with thestructure. 1-Azidotriethylene glycol (compound 224, 5.53 g, 23.69 mmol)and compound 4 (6 g, 18.22 mmol) were treated with 4 A molecular sieves(5 g), and TMSOTf (1.65 ml, 9.11 mmol) in dichloromethane (100 mL) underan inert atmosphere. After 14 hours, the reaction was filtered to removethe sieves, and the organic layer was washed with sat. NaHCO₃, water,brine, and dried over Na₂SO₄. The organic layer was concentrated todryness and purified by column chromatography, eluted with a gradient of2 to 4% methanol in dichloromethane to give compound 225. LCMS and NMRwere consistent with the structure. Compound 225 (11.9 g, 23.59 mmol)was hydrogenated in EtOAc/Methanol (4:1, 250 mL) over Pearlman'scatalyst. After 8 hours, the catalyst was removed by filtration and thesolvents removed to dryness to give compound 226. LCMS and NMR wereconsistent with the structure.

In order to generate compound 227, a solution ofnitromethanetrispropionic acid (4.17 g, 15.04 mmol) and Hunig's base(10.3 ml, 60.17 mmol) in DMF (100 mL) were treated dropwise withpentaflourotrifluoro acetate (9.05 ml, 52.65 mmol). After 30 minutes,the reaction was poured onto ice water and extracted with EtOAc. Theorganic layer was washed with water, brine, and dried over Na₂SO₄. Theorganic layer was concentrated to dryness and then recrystallized fromheptane to give compound 227 as a white solid. LCMS and NMR wereconsistent with the structure. Compound 227 (1.5 g, 1.93 mmol) andcompound 226 (3.7 g, 7.74 mmol) were stirred at room temperature inacetonitrile (15 mL) for 2 hours. The reaction was then evaporated todryness and purified by column chromatography, eluting with a gradientof 2 to 10% methanol in dichloromethane to give compound 228. LCMS andNMR were consistent with the structure. Compound 228 (1.7 g, 1.02 mmol)was treated with Raney Nickel (about 2 g wet) in ethanol (100 mL) in anatmosphere of hydrogen. After 12 hours, the catalyst was removed byfiltration and the organic layer was evaporated to a solid that was useddirectly in the next step. LCMS and NMR were consistent with thestructure. This solid (0.87 g, 0.53 mmol) was treated withbenzylglutaric acid (0.18 g, 0.8 mmol), HBTU (0.3 g, 0.8 mmol) and DIEA(273.7 μl, 1.6 mmol) in DMF (5 mL). After 16 hours, the DMF was removedunder reduced pressure at 65° C. to an oil, and the oil was dissolved indichloromethane. The organic layer was washed with sat. NaHCO₃, brine,and dried over Na₂SO₄. After evaporation of the organic layer, thecompound was purified by column chromatography and eluted with agradient of 2 to 20% methanol in dichloromethane to give the coupledproduct. LCMS and NMR were consistent with the structure. The benzylester was deprotected with Pearlman's catalyst under a hydrogenatmosphere for 1 hour. The catalyst was them removed by filtration andthe solvents removed to dryness to give the acid. LCMS and NMR wereconsistent with the structure. The acid (486 mg, 0.27 mmol) wasdissolved in dry DMF (3 mL). Pyridine (53.61 μl, 0.66 mmol) was addedand the reaction was purged with argon. Pentaflourotriflouro acetate(46.39 μl, 0.4 mmol) was slowly added to the reaction mixture. The colorof the reaction changed from pale yellow to burgundy, and gave off alight smoke which was blown away with a stream of argon. The reactionwas allowed to stir at room temperature for one hour (completion ofreaction was confirmed by LCMS). The solvent was removed under reducedpressure (rotovap) at 70° C. The residue was diluted with DCM and washedwith 1N NaHSO₄, brine, saturated sodium bicarbonate and brine again. Theorganics were dried over Na₂SO₄, filtered, and were concentrated todryness to give 225 mg of compound 229 as a brittle yellow foam. LCMSand NMR were consistent with the structure.

Oligomeric compound 230, comprising a GalNAc₃-23 conjugate group, wasprepared from compound 229 using the general procedure illustrated inExample 46. The GalNAc₃ cluster portion of the GalNAc₃-23 conjugategroup (GalNAc₃-23_(a)) can be combined with any cleavable moiety toprovide a variety of conjugate groups. The structure of GalNAc₃-23(GalNAc₃-23_(a)-CM) is shown below:

Example 77: Antisense Inhibition In Vivo by Oligonucleotides TargetingSRB-1 Comprising a GalNAc₃ Conjugate

The oligonucleotides listed below were tested in a dose-dependent studyfor antisense inhibition of SRB-1 in mice.

TABLE 64 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 661161 GalNAc ₃ -3 _(a) - _(o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a A_(d) 4888 G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 666904 GalNAc ₃-3 _(a) - _(o′)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a PO 4886G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)673502 GalNAc ₃ -10 _(a) - _(o′) A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-10a A_(d)4888 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo)^(m)C_(es)T_(es)T_(e) 677844 GalNAc ₃ -9 _(a) - _(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-9a A_(d) 4888 G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 677843 GalNAc ₃-23 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-23a A_(d)4888 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) 655861 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) GalNAc₃-1a A_(d) 4887^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a) 677841 G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)GalNAc₃-19a A_(d) 4887 ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -19_(a) 677842 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)GalNAc₃-20a A_(d) 4887 ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -20_(a)

The structure of GalNAc₃-1_(a) was shown previously in Example 9,GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-9a was shown in Example52, GalNAc₃-10a was shown in Example 46, GalNAc₃-19_(a) was shown inExample 70, GalNAc₃-20_(a) was shown in Example 71, and GalNAc₃-23_(a)was shown in Example 76.

Treatment

Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.)were each injected subcutaneously once at a dosage shown below with anoligonucleotide listed in Table 64 or with saline. Each treatment groupconsisted of 4 animals. The mice were sacrificed 72 hours following thefinal administration to determine the SRB-1 mRNA levels using real-timePCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc.Eugene, Oreg.) according to standard protocols. The results below arepresented as the average percent of SRB-1 mRNA levels for each treatmentgroup, normalized to the saline control.

As illustrated in Table 65, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner.

TABLE 65 SRB-1 mRNA (% Saline) Dosage SRB-1 mRNA GalNAc₃ ISIS No.(mg/kg) (% Saline) Cluster CM Saline n/a 100.0 n/a n/a 661161 0.5 89.18GalNAc₃-3a A_(d) 1.5 77.02 5 29.10 15 12.64 666904 0.5 93.11 GalNAc₃-3aPO 1.5 55.85 5 21.29 15 13.43 673502 0.5 77.75 GalNAc₃-10a A_(d) 1.541.05 5 19.27 15 14.41 677844 0.5 87.65 GalNAc₃-9a A_(d) 1.5 93.04 540.77 15 16.95 677843 0.5 102.28 GalNAc₃-23a A_(d) 1.5 70.51 5 30.68 1513.26 655861 0.5 79.72 GalNAc₃-1a A_(d) 1.5 55.48 5 26.99 15 17.58677841 0.5 67.43 GalNAc₃-19a A_(d) 1.5 45.13 5 27.02 15 12.41 677842 0.564.13 GalNAc₃-20a A_(d) 1.5 53.56 5 20.47 15 10.23

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were also measured using standardprotocols. Total bilirubin and BUN were also evaluated. Changes in bodyweights were evaluated, with no significant change from the saline group(data not shown). ALTs, ASTs, total bilirubin and BUN values are shownin Table 66 below.

TABLE 66 Total Dosage ALT AST Bilirubin BUN GalNAc₃ ISIS No. (mg/kg)(U/L) (U/L) (mg/dL) (mg/dL) Cluster CM Saline n/a 21 45 0.13 34 n/a n/a661161 0.5 28 51 0.14 39 GalNAc₃-3a A_(d) 1.5 23 42 0.13 39 5 22 59 0.1337 15 21 56 0.15 35 666904 0.5 24 56 0.14 37 GalNAc₃-3a PO 1.5 26 680.15 35 5 23 77 0.14 34 15 24 60 0.13 35 673502 0.5 24 59 0.16 34GalNAc₃-10a A_(d) 1.5 20 46 0.17 32 5 24 45 0.12 31 15 24 47 0.13 34677844 0.5 25 61 0.14 37 GalNAc₃-9a A_(d) 1.5 23 64 0.17 33 5 25 58 0.1335 15 22 65 0.14 34 677843 0.5 53 53 0.13 35 GalNAc₃-23a A_(d) 1.5 25 540.13 34 5 21 60 0.15 34 15 22 43 0.12 38 655861 0.5 21 48 0.15 33GalNAc₃-1a A_(d) 1.5 28 54 0.12 35 5 22 60 0.13 36 15 21 55 0.17 30677841 0.5 32 54 0.13 34 GalNAc₃-19a A_(d) 1.5 24 56 0.14 34 5 23 920.18 31 15 24 58 0.15 31 677842 0.5 23 61 0.15 35 GalNAc₃-20a A_(d) 1.524 57 0.14 34 5 41 62 0.15 35 15 24 37 0.14 32

Example 78: Antisense Inhibition In Vivo by Oligonucleotides TargetingAngiotensinogen Comprising a GalNAc₃ Conjugate

The oligonucleotides listed below were tested in a dose-dependent studyfor antisense inhibition of Angiotensinogen (AGT) in normotensiveSprague Dawley rats.

TABLE 67 Modified ASOs targeting AGT ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 552668 ^(m)C_(es)A_(es)^(m)C_(es)T_(es)G_(es)A_(ds) n/a n/a 4892T_(ds)T_(ds)T_(ds)T_(ds)T_(ds)G_(ds) ^(m)C_(ds) ^(m)C_(ds)^(m)C_(ds)A_(es)G_(es)G_(es) A_(es)T_(e) 669509 ^(m)C_(es)A_(es)^(m)C_(es)T_(es)G_(es)A_(ds) GalNAc₃-1_(a) A_(d) 4893T_(ds)T_(ds)T_(ds)T_(ds)T_(ds)G_(ds) ^(m)C_(ds) ^(m)C_(ds)^(m)C_(ds)A_(es)G_(es)G_(es) A_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a)

The structure of GalNAc₃-1a was shown previously in Example 9.

Treatment

Six week old, male Sprague Dawley rats were each injected subcutaneouslyonce per week at a dosage shown below, for a total of three doses, withan oligonucleotide listed in Table 67 or with PBS. Each treatment groupconsisted of 4 animals. The rats were sacrificed 72 hours following thefinal dose. AGT liver mRNA levels were measured using real-time PCR andRIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene,Oreg.) according to standard protocols. AGT plasma protein levels weremeasured using the Total Angiotensinogen ELISA (Catalog # JP27412, IBLInternational, Toronto, ON) with plasma diluted 1:20,000. The resultsbelow are presented as the average percent of AGT mRNA levels in liveror AGT protein levels in plasma for each treatment group, normalized tothe PBS control.

As illustrated in Table 68, treatment with antisense oligonucleotideslowered AGT liver mRNA and plasma protein levels in a dose-dependentmanner, and the oligonucleotide comprising a GalNAc conjugate wassignificantly more potent than the parent oligonucleotide lacking aGalNAc conjugate.

TABLE 68 AGT liver mRNA and plasma protein levels AGT AGT liver plasmaDosage mRNA protein GalNAc₃ ISIS No. (mg/kg) (% PBS) (% PBS) Cluster CMPBS n/a 100 100 n/a n/a 552668 3 95 122 n/a n/a 10 85 97 30 46 79 90 811 669509 0.3 95 70 GalNAc₃-1a A_(d) 1 95 129 3 62 97 10 9 23

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in plasma and body weights were also measured attime of sacrifice using standard protocols. The results are shown inTable 69 below.

TABLE 69 Liver transaminase levels and rat body weights Body Dosage ALTAST Weight GalNAc₃ ISIS No. (mg/kg) (U/L) (U/L) (% of baseline) ClusterCM PBS n/a 51 81 186 n/a n/a 552668 3 54 93 183 n/a n/a 10 51 93 194 3059 99 182 90 56 78 170 669509 0.3 53 90 190 GalNAc₃-1a A_(d) 1 51 93 1923 48 85 189 10 56 95 189

Example 79: Duration of Action In Vivo of Oligonucleotides TargetingAPOC-III Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 70 below were tested in a singledose study for duration of action in mice.

TABLE 70 Modified ASOs targeting APOC-III ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 304801 A_(es)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds)^(m)C_(ds)T_(es)T_(es) n/a n/a 4878 T_(es)A_(es)T_(e) 647535A_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds)^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es) GalNAc₃-1aA_(d) 4879 T_(es)A_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a) 663083 GalNAc ₃-3 _(a) - _(o′) A _(do)A_(es)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) GalNAc₃-3a A_(d) 4894^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) 674449GalNAC ₃ -7 _(a) - _(o′) A _(do)A_(es)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) GalNAc₃-7a A_(d) 4894^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) 674450GalNAc ₃ -10 _(a) - _(o′) A _(do)A_(es)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) GalNAc₃-10a A_(d) 4894^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) 674451GalNAc ₃ -13 _(a) - _(o′) A _(do)A_(es)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) GalNAc₃-13a A_(d) 4894^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e)The structure of GalNAc₃-1_(a) was shown previously in Example 9,GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-7_(a) was shown inExample 48, GalNAc₃-10_(a) was shown in Example 46, and GalNAc₃-13_(a)was shown in Example 62.

Treatment

Six to eight week old transgenic mice that express human APOC-III wereeach injected subcutaneously once with an oligonucleotide listed inTable 70 or with PBS. Each treatment group consisted of 3 animals. Bloodwas drawn before dosing to determine baseline and at 72 hours, 1 week, 2weeks, 3 weeks, 4 weeks, 5 weeks, and 6 weeks following the dose. Plasmatriglyceride and APOC-III protein levels were measured as described inExample 20. The results below are presented as the average percent ofplasma triglyceride and APOC-III levels for each treatment group,normalized to baseline levels, showing that the oligonucleotidescomprising a GalNAc conjugate group exhibited a longer duration ofaction than the parent oligonucleotide without a conjugate group (ISIS304801) even though the dosage of the parent was three times the dosageof the oligonucleotides comprising a GalNAc conjugate group.

TABLE 71 Plasma triglyceride and APOC-III protein levels in transgenicmice Time point APOC-III Dosage (days post- Triglycerides proteinGalNAc₃ ISIS No. (mg/kg) dose) (% baseline) (% baseline) Cluster CM PBSn/a 3 97 102 n/a n/a 7 101 98 14 108 98 21 107 107 28 94 91 35 88 90 4291 105 304801 30 3 40 34 n/a n/a 7 41 37 14 50 57 21 50 50 28 57 73 3568 70 42 75 93 647535 10 3 36 37 GalNAc₃-1a A_(d) 7 39 47 14 40 45 21 4141 28 42 62 35 69 69 42 85 102 663083 10 3 24 18 GalNAc₃-3a A_(d) 7 2823 14 25 27 21 28 28 28 37 44 35 55 57 42 60 78 674449 10 3 29 26GalNAc₃-7a A_(d) 7 32 31 14 38 41 21 44 44 28 53 63 35 69 77 42 78 99674450 10 3 33 30 GalNAc₃-10a A_(d) 7 35 34 14 31 34 21 44 44 28 56 6135 68 70 42 83 95 674451 10 3 35 33 GalNAc₃-13a A_(d) 7 24 32 14 40 3421 48 48 28 54 67 35 65 75 42 74 97

Example 80: Antisense Inhibition In Vivo by Oligonucleotides TargetingAlpha-1 Antitrypsin (A1AT) Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 72 below were tested in a study fordose-dependent inhibition of A1AT in mice.

TABLE 72 Modified ASOs targeting A1AT ISIS GalNAc₃ SEQ ID No. Sequences(5′ to 3′) Cluster CM No. 476366 A_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(ds)G_(ds)A_(ds)A_(ds)G_(ds)G_(ds)A_(es)A_(es) n/a n/a 4895G_(es)G_(es)A_(e) 656326 A_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(ds)G_(ds)A_(ds)A_(ds)G_(ds)G_(ds)A_(es)A_(es) GalNAc₃-1aA_(d) 4896 G_(es)G_(es)A_(eo) A _(do′) -GalNAc ₃ -1 _(a) 678381 GalNAc ₃-3 _(a) - _(o′) A _(do)A_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds)A_(ds)GalNAc₃-3a A_(d) 4897 A_(ds)G_(ds)G_(ds)A_(es)A_(es)G_(es)G_(es)A_(e)678382 GalNAc ₃ -7 _(a) - _(o′) A _(do)A_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds)A_(ds)GalNAc₃-7a A_(d) 4897 A_(ds)G_(ds)G_(ds)A_(es)A_(es)G_(es)G_(es)A_(e)678383 GalNAc ₃ -10 _(a) - _(o′) A _(do)A_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds) GalNAc₃-10aA_(d) 4897 A_(ds)A_(ds)G_(ds)G_(ds)A_(es)A_(es)G_(es)G_(es)A_(e) 678384GalNAc ₃ -13 _(a) - _(o′) A _(do)A_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds) GalNAc₃-13aA_(d) 4897 A_(ds)A_(ds)G_(ds)G_(ds)A_(es)A_(es)G_(es)G_(es)A_(e)

The structure of GalNAc₃-1_(a) was shown previously in Example 9,GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-7_(a) was shown inExample 48, GalNAc₃-10_(a) was shown in Example 46, and GalNAc₃-13_(a)was shown in Example 62.

Treatment

Six week old, male C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.)were each injected subcutaneously once per week at a dosage shown below,for a total of three doses, with an oligonucleotide listed in Table 72or with PBS. Each treatment group consisted of 4 animals. The mice weresacrificed 72 hours following the final administration. A1AT liver mRNAlevels were determined using real-time PCR and RIBOGREEN RNAquantification reagent (Molecular Probes, Inc. Eugene, Oreg.) accordingto standard protocols. A1AT plasma protein levels were determined usingthe Mouse Alpha 1-Antitrypsin ELISA (catalog #41-A1AMS-E01, Alpco,Salem, N.H.). The results below are presented as the average percent ofA1AT liver mRNA and plasma protein levels for each treatment group,normalized to the PBS control.

As illustrated in Table 73, treatment with antisense oligonucleotideslowered A1AT liver mRNA and A1AT plasma protein levels in adose-dependent manner. The oligonucleotides comprising a GalNAcconjugate were significantly more potent than the parent (ISIS 476366).

TABLE 73 A1AT liver mRNA and plasma protein levels A1AT A1AT liverplasma Dosage mRNA protein GalNAc₃ ISIS No. (mg/kg) (% PBS) (% PBS)Cluster CM PBS n/a 100 100 n/a n/a 476366 5 86 78 n/a n/a 15 73 61 45 3038 656326 0.6 99 90 GalNAc₃-1a A_(d) 2 61 70 6 15 30 18 6 10 678381 0.6105 90 GalNAc₃-3a A_(d) 2 53 60 6 16 20 18 7 13 678382 0.6 90 79GalNAc₃-7a A_(d) 2 49 57 6 21 27 18 8 11 678383 0.6 94 84 GalNAc₃-10aA_(d) 2 44 53 6 13 24 18 6 10 678384 0.6 106 91 GalNAc₃-13a A_(d) 2 6559 6 26 31 18 11 15

Liver transaminase and BUN levels in plasma were measured at time ofsacrifice using standard protocols. Body weights and organ weights werealso measured. The results are shown in Table 74 below. Body weight isshown as % relative to baseline. Organ weights are shown as % of bodyweight relative to the PBS control group.

TABLE 74 Body Liver Kidney Spleen Dosage ALT AST BUN weight weightweight weight ISIS No. (mg/kg) (U/L) (U/L) (mg/dL) (% baseline) (Rel %BW) (Rel % BW) (Rel % BW) PBS n/a 25 51 37 119 100 100 100 476366 5 3468 35 116 91 98 106 15 37 74 30 122 92 101 128 45 30 47 31 118 99 108123 656326 0.6 29 57 40 123 100 103 119 2 36 75 39 114 98 111 106 6 3267 39 125 99 97 122 18 46 77 36 116 102 109 101 678381 0.6 26 57 32 11793 109 110 2 26 52 33 121 96 106 125 6 40 78 32 124 92 106 126 18 31 5428 118 94 103 120 678382 0.6 26 42 35 114 100 103 103 2 25 50 31 117 91104 117 6 30 79 29 117 89 102 107 18 65 112 31 120 89 104 113 678383 0.630 67 38 121 91 100 123 2 33 53 33 118 98 102 121 6 32 63 32 117 97 105105 18 36 68 31 118 99 103 108 678384 0.6 36 63 31 118 98 103 98 2 32 6132 119 93 102 114 6 34 69 34 122 100 100 96 18 28 54 30 117 98 101 104

Example 81: Duration of Action In Vivo of Oligonucleotides TargetingA1AT Comprising a GalNAc₃ Cluster

The oligonucleotides listed in Table 72 were tested in a single dosestudy for duration of action in mice.

Treatment

Six week old, male C57BL/6 mice were each injected subcutaneously oncewith an oligonucleotide listed in Table 72 or with PBS. Each treatmentgroup consisted of 4 animals. Blood was drawn the day before dosing todetermine baseline and at 5, 12, 19, and 25 days following the dose.Plasma A1AT protein levels were measured via ELISA (see Example 80). Theresults below are presented as the average percent of plasma A1ATprotein levels for each treatment group, normalized to baseline levels.The results show that the oligonucleotides comprising a GalNAc conjugatewere more potent and had longer duration of action than the parentlacking a GalNAc conjugate (ISIS 476366). Furthermore, theoligonucleotides comprising a 5′-GalNAc conjugate (ISIS 678381, 678382,678383, and 678384) were generally even more potent with even longerduration of action than the oligonucleotide comprising a 3′-GalNAcconjugate (ISIS 656326).

TABLE 75 Plasma A1AT protein levels in mice Time point Dosage (dayspost- A1AT GalNAc₃ ISIS No. (mg/kg) dose) (% baseline) Cluster CM PBSn/a 5 93 n/a n/a 12 93 19 90 25 97 476366 100 5 38 n/a n/a 12 46 19 6225 77 656326 18 5 33 GalNAc₃-1a A_(d) 12 36 19 51 25 72 678381 18 5 21GalNAc₃-3a A_(d) 12 21 19 35 25 48 678382 18 5 21 GalNAc₃-7a A_(d) 12 2119 39 25 60 678383 18 5 24 GalNAc₃-10a A_(d) 12 21 19 45 25 73 678384 185 29 GalNAc₃-13a A_(d) 12 34 19 57 25 76

Example 82: Antisense Inhibition In Vitro by Oligonucleotides TargetingSRB-1 Comprising a GalNAc₃ Conjugate

Primary mouse liver hepatocytes were seeded in 96 well plates at 15,000cells/well 2 hours prior to treatment. The oligonucleotides listed inTable 76 were added at 2, 10, 50, or 250 nM in Williams E medium andcells were incubated overnight at 37° C. in 5% CO₂. Cells were lysed 16hours following oligonucleotide addition, and total RNA was purifiedusing RNease 3000 BioRobot (Qiagen). SRB-1 mRNA levels were determinedusing real-time PCR and RIBOGREEN RNA quantification reagent (MolecularProbes, Inc. Eugene, Oreg.) according to standard protocols. IC₅₀ valueswere determined using Prism 4 software (GraphPad). The results show thatoligonucleotides comprising a variety of different GalNAc conjugategroups and a variety of different cleavable moieties are significantlymore potent in an in vitro free uptake experiment than the parentoligonucleotides lacking a GalNAc conjugate group (ISIS 353382 and666841).

TABLE 76 Inhibition of SRB-1 expression in vitro ISIS GalNAc IC₅₀ SEQNo. Sequence (5′ to 3′) Linkages cluster CM (nM) ID No. 353382 G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PS n/a n/a 250 4886^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 655861 G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PS GalNAc₃- A_(d) 40 4887^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′)-GalNAC ₃ -1a 1_(a) 661161 GalNAc ₃ -3 _(a) - _(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 404888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es)^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 3_(a) 661162 GalNAc ₃ -3 _(a) - _(o′) A_(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PSGalNAc₃- A_(d) 8 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 3_(a) 664078G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PS GalNAc₃- A_(d) 20 4887^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′)-GalNAc ₃ -9 _(a) 9_(a) 665001 GalNAc ₃ -8 _(a) - _(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 704888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es)^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 8_(a) 666224 GalNAc ₃ -5 _(a) - _(o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PSGalNAc₃- A_(d) 80 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 5_(a) 666841G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PO/PS n/a n/a >250 4886^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 666881 GalNAc ₃-10 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 30 4888^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) 10_(a) 666904 GalNAc ₃ -3 _(a) - _(o′)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds) PSGalNAc₃- PO 9 4886 A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds T) _(es)^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 3_(a) 666924 GalNAc ₃ -3 _(a) - _(o′) T_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PSGalNAc₃- T_(d) 15 4891 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 3_(a) 666961GalNAC ₃ -6 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 150 4888^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) 6_(a) 666981 GalNAC ₃ -7 _(a) - _(o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PSGalNAc₃- A_(d) 20 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 7_(a) 670061GalNAC ₃ -13 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 30 4888^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) 13_(a) 670699 GalNAC ₃ -3 _(a) - _(o′) T_(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PSGalNAc₃- T_(d) 30 4891 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 3_(a) 670700GalNAC ₃ -3 _(a) - _(o′) A _(eo)G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- A_(e) 30 4888^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo)^(m)C_(es)T_(es)T 3_(a) 670701 GalNAC ₃ -3 _(a) - _(o′) T _(eo)G_(es)^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- T_(e)25 4891 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo)^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 3_(a) 671144 GalNAC ₃ -12 _(a) - _(o′)A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PSGalNAc₃- A_(d) 30 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 12_(a) 671165GalNAC ₃ -13 _(a) - _(o′) A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- A_(d) 8 4888^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo)^(m)C_(es)T_(es)T 13_(a) 671261 GalNAC ₃ -14 _(a) - _(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃-A_(d) >250 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 14_(a) 671262GalNAC ₃ -15 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) >250 4888^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) 15_(a) 673501 GalNAC ₃ -7 _(a) - _(o′) A_(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PSGalNAc₃- A_(d) 30 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 7_(a) 673502GalNAC ₃ -10 _(a) - _(o′) A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- A_(d) 8 4888^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo)^(m)C_(es)T_(es)T_(e) 10_(a) 675441 GalNAC ₃ -17 _(a) - _(o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PSGalNAc₃- A_(d) 30 4888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 17_(a) 675442GalNAC ₃ -18 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 20 4888^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) 18_(a) 677841 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PSGalNAc₃- A_(d) 40 4887 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAC ₃ -19 _(a) 19_(a) 677842 G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PS GalNAc₃- A_(d) 30 4887^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′)-GalNAC ₃ -20 _(a) 20_(a) 677843 GalNAC ₃ -23 _(a) - _(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 404888 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es)^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 23_(a)The structure of GalNAc₃-1_(a) was shown previously in Example 9,GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-5_(a) was shown inExample 49, GalNAc₃-6_(a) was shown in Example 51, GalNAc₃-7_(a) wasshown in Example 48, GalNAc₃-8_(a) was shown in Example 47,GalNAc₃-9_(a) was shown in Example 52, GalNAc₃-10_(a) was shown inExample 46, GalNAc₃-12_(a) was shown in Example 61, GalNAc₃-13_(a) wasshown in Example 62, GalNAc₃-14_(a) was shown in Example 63,GalNAc₃-15_(a) was shown in Example 64, GalNAc₃-17_(a) was shown inExample 68, GalNAc₃-18_(a) was shown in Example 69, GalNAc₃-19_(a) wasshown in Example 70, GalNAc₃-20_(a) was shown in Example 71, andGalNAc₃-23_(a) was shown in Example 76.

Example 83: Antisense Inhibition In Vivo by Oligonucleotides TargetingFactor XI Comprising a GalNAc₃ Cluster

The oligonucleotides listed in Table 77 below were tested in a study fordose-dependent inhibition of Factor XI in mice.

TABLE 77 Modified oligonucleotides targeting Factor XI ISIS GalNAc SEQNo. Sequence (5′ to 3′) cluster CM ID No. 404071T_(es)G_(es)G_(es)T_(es)A_(es)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(es)G_(es) n/a n/a 4889A_(es)G_(es)Ge 656173 T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds)^(m)C_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(eo)G_(eo) GalNAc₃-1_(a) A_(d) 4890 A_(es)G_(es)G_(eo) A_(do′) -GalNAC ₃ -1 _(a) 663086 GalNAc ₃ -3 _(a) - _(o′) A_(do)T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds) ^(m)C_(ds)T_(ds) GalNAc₃-3_(a) A_(d) 4898 T_(ds)T_(ds)^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(e) 678347 GalNAc ₃ -7 _(a) - _(o′)A _(do)T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds) ^(m)C_(ds)T_(ds) GalNAc₃-7_(a) A_(d) 4898 T_(ds)T_(ds)^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(e) 678348 GalNAc ₃ -10 _(a) - _(o′)A _(do)T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-10_(a) A_(d) 4898 T_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(e) 678349 GalNAc ₃ -13 _(a) - _(o′)A _(do)T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-13_(a) A_(d) 4898 T_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(e)

The structure of GalNAc₃-1_(a) was shown previously in Example 9,GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-7_(a) was shown inExample 48, GalNAc₃-10_(a) was shown in Example 46, and GalNAc₃-13_(a)was shown in Example 62.

Treatment

Six to eight week old mice were each injected subcutaneously once perweek at a dosage shown below, for a total of three doses, with anoligonucleotide listed below or with PBS. Each treatment group consistedof 4 animals. The mice were sacrificed 72 hours following the finaldose. Factor XI liver mRNA levels were measured using real-time PCR andnormalized to cyclophilin according to standard protocols. Livertransaminases, BUN, and bilirubin were also measured. The results beloware presented as the average percent for each treatment group,normalized to the PBS control.

As illustrated in Table 78, treatment with antisense oligonucleotideslowered Factor XI liver mRNA in a dose-dependent manner. The resultsshow that the oligonucleotides comprising a GalNAc conjugate were morepotent than the parent lacking a GalNAc conjugate (ISIS 404071).Furthermore, the oligonucleotides comprising a 5′-GalNAc conjugate (ISIS663086, 678347, 678348, and 678349) were even more potent than theoligonucleotide comprising a 3′-GalNAc conjugate (ISIS 656173).

TABLE 78 Factor XI liver mRNA, liver transaminase, BUN, and bilirubinlevels Factor XI Dosage mRNA ALT AST BUN Bilirubin GalNAc₃ SEQ ISIS No.(mg/kg) (% PBS) (U/L) (U/L) (mg/dL) (mg/dL) Cluster ID No. PBS n/a 10063 70 21 0.18 n/a n/a 404071 3 65 41 58 21 0.15 n/a 4889 10 33 49 53 230.15 30 17 43 57 22 0.14 656173 0.7 43 90 89 21 0.16 GalNAc₃-1a 4890 2 936 58 26 0.17 6 3 50 63 25 0.15 663086 0.7 33 91 169 25 0.16 GalNAc₃-3a4898 2 7 38 55 21 0.16 6 1 34 40 23 0.14 678347 0.7 35 28 49 20 0.14GalNAc₃-7a 4898 2 10 180 149 21 0.18 6 1 44 76 19 0.15 678348 0.7 39 4354 21 0.16 GalNAc₃-10a 4898 2 5 38 55 22 0.17 6 2 25 38 20 0.14 6783490.7 34 39 46 20 0.16 GalNAc₃-13a 4898 2 8 43 63 21 0.14 6 2 28 41 200.14

Example 84: Duration of Action In Vivo of Oligonucleotides TargetingFactor XI Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 77 were tested in a single dosestudy for duration of action in mice.

Treatment Six to eight week old mice were each injected subcutaneouslyonce with an oligonucleotide listed in Table 77 or with PBS. Eachtreatment group consisted of 4 animals. Blood was drawn by tail bleedsthe day before dosing to determine baseline and at 3, 10, and 17 daysfollowing the dose. Plasma Factor XI protein levels were measured byELISA using Factor XI capture and biotinylated detection antibodies fromR & D Systems, Minneapolis, Minn. (catalog # AF2460 and # BAF2460,respectively) and the OptEIA Reagent Set B (Catalog #550534, BDBiosciences, San Jose, Calif.). The results below are presented as theaverage percent of plasma Factor XI protein levels for each treatmentgroup, normalized to baseline levels. The results show that theoligonucleotides comprising a GalNAc conjugate were more potent withlonger duration of action than the parent lacking a GalNAc conjugate(ISIS 404071). Furthermore, the oligonucleotides comprising a 5′-GalNAcconjugate (ISIS 663086, 678347, 678348, and 678349) were even morepotent with an even longer duration of action than the oligonucleotidecomprising a 3′-GalNAc conjugate (ISIS 656173).

TABLE 79 Plasma Factor XI protein levels in mice ISIS Dosage Time point(days Factor XI (% SEQ ID No. (mg/kg) post-dose) baseline) GalNAc₃Cluster CM No. PBS n/a 3 123 n/a n/a n/a 10 56 17 100 404071 30 3 11 n/an/a 4889 10 47 17 52 656173 6 3 1 GalNAc₃-1a A_(d) 4890 10 3 17 21663086 6 3 1 GalNAc₃-3a A_(d) 4898 10 2 17 9 678347 6 3 1 GalNAc₃-7aA_(d) 4898 10 1 17 8 678348 6 3 1 GalNAc₃-10a A_(d) 4898 10 1 17 6678349 6 3 1 GalNAc₃-13a A_(d) 4898 10 1 17 5

Example 85: Antisense Inhibition In Vivo by Oligonucleotides TargetingSRB-1 Comprising a GalNAc₃ Conjugate

Oligonucleotides listed in Table 76 were tested in a dose-dependentstudy for antisense inhibition of SRB-1 in mice.

Treatment

Six to eight week old C57BL/6 mice were each injected subcutaneouslyonce per week at a dosage shown below, for a total of three doses, withan oligonucleotide listed in Table 76 or with saline. Each treatmentgroup consisted of 4 animals. The mice were sacrificed 48 hoursfollowing the final administration to determine the SRB-1 mRNA levelsusing real-time PCR and RIBOGREEN® RNA quantification reagent (MolecularProbes, Inc. Eugene, Oreg.) according to standard protocols. The resultsbelow are presented as the average percent of liver SRB-1 mRNA levelsfor each treatment group, normalized to the saline control.

As illustrated in Tables 80 and 81, treatment with antisenseoligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner.

TABLE 80 SRB-1 mRNA in liver Dosage SRB-1 mRNA GalNAc₃ ISIS No. (mg/kg)(% Saline) Cluster CM Saline n/a 100 n/a n/a 655861 0.1 94 GalNAc₃-1aA_(d) 0.3 119 1 68 3 32 661161 0.1 120 GalNAc₃-3a A_(d) 0.3 107 1 68 326 666881 0.1 107 GalNAc₃-10a A_(d) 0.3 107 1 69 3 27 666981 0.1 120GalNAc₃-7a A_(d) 0.3 103 1 54 3 21 670061 0.1 118 GalNAc₃-13a A_(d) 0.389 1 52 3 18 677842 0.1 119 GalNAc₃-20a A_(d) 0.3 96 1 65 3 23

TABLE 81 SRB-1 mRNA in liver Dosage SRB-1 mRNA GalNAc₃ ISIS No. (mg/kg)(% Saline) Cluster CM 661161 0.1 107 GalNAc₃-3a A_(d) 0.3 95 1 53 3 18677841 0.1 110 GalNAc₃-19a A_(d) 0.3 88 1 52 3 25

Liver transaminase levels, total bilirubin, BUN, and body weights werealso measured using standard protocols. Average values for eachtreatment group are shown in Table 82 below.

TABLE 82 ISIS Dosage ALT AST Bilirubin BUN Body Weight No. (mg/kg) (U/L)(U/L) (mg/dL) (mg/dL) (% baseline) GalNAc₃ Cluster CM Saline n/a 19 390.17 26 118 n/a n/a 655861 0.1 25 47 0.17 27 114 GalNAc₃-1a A_(d) 0.3 2956 0.15 27 118 1 20 32 0.14 24 112 3 27 54 0.14 24 115 661161 0.1 35 830.13 24 113 GalNAc₃-3a A_(d) 0.3 42 61 0.15 23 117 1 34 60 0.18 22 116 329 52 0.13 25 117 666881 0.1 30 51 0.15 23 118 GalNAc₃-10a A_(d) 0.3 4982 0.16 25 119 1 23 45 0.14 24 117 3 20 38 0.15 21 112 666981 0.1 21 410.14 22 113 GalNAc₃-7a A_(d) 0.3 29 49 0.16 24 112 1 19 34 0.15 22 111 377 78 0.18 25 115 670061 0.1 20 63 0.18 24 111 GalNAc₃-13a A_(d) 0.3 2057 0.15 21 115 1 20 35 0.14 20 115 3 27 42 0.12 20 116 677842 0.1 20 380.17 24 114 GalNAc₃-20a A_(d) 0.3 31 46 0.17 21 117 1 22 34 0.15 21 1193 41 57 0.14 23 118

Example 86: Antisense Inhibition In Vivo by Oligonucleotides TargetingTTR Comprising a GalNAc₃ Cluster

Oligonucleotides listed in Table 83 below were tested in adose-dependent study for antisense inhibition of human transthyretin(TTR) in transgenic mice that express the human TTR gene.

Treatment

Eight week old TTR transgenic mice were each injected subcutaneouslyonce per week for three weeks, for a total of three doses, with anoligonucleotide and dosage listed in the tables below or with PBS. Eachtreatment group consisted of 4 animals. The mice were sacrificed 72hours following the final administration. Tail bleeds were performed atvarious time points throughout the experiment, and plasma TTR protein,ALT, and AST levels were measured and reported in Tables 85-87. Afterthe animals were sacrificed, plasma ALT, AST, and human TTR levels weremeasured, as were body weights, organ weights, and liver human TTR mRNAlevels. TTR protein levels were measured using a clinical analyzer(AU480, Beckman Coulter, CA). Real-time PCR and RIBOGREEN RNAquantification reagent (Molecular Probes, Inc. Eugene, Oreg.) were usedaccording to standard protocols to determine liver human TTR mRNAlevels. The results presented in Tables 84-87 are the average values foreach treatment group. The mRNA levels are the average values relative tothe average for the PBS group. Plasma protein levels are the averagevalues relative to the average value for the PBS group at baseline. Bodyweights are the average percent weight change from baseline untilsacrifice for each individual treatment group. Organ weights shown arenormalized to the animal's body weight, and the average normalized organweight for each treatment group is then presented relative to theaverage normalized organ weight for the PBS group.

In Tables 84-87, “BL” indicates baseline, measurements that were takenjust prior to the first dose. As illustrated in Tables 84 and 85,treatment with antisense oligonucleotides lowered TTR expression levelsin a dose-dependent manner. The oligonucleotides comprising a GalNAcconjugate were more potent than the parent lacking a GalNAc conjugate(ISIS 420915). Furthermore, the oligonucleotides comprising a GalNAcconjugate and mixed PS/PO internucleoside linkages were even more potentthan the oligonucleotide comprising a GalNAc conjugate and full PSlinkages.

TABLE 83 Oligonucleotides targeting human TTR GalNAc SEQ Isis No.Sequence 5′ to 3′ Linkages cluster CM ID No. 420915 T_(es)^(m)C_(es)T_(es)T_(es)G_(es)G_(ds)T_(ds)T_(ds)A_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds) PS n/a n/a 4899 A_(es)T_(es)^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 660261 T_(es)^(m)C_(es)T_(es)T_(es)G_(es)G_(ds)T_(ds)T_(ds)A_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds) PS GalNAc₃-1a A_(d) 4900A_(es)T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(eo) A _(do′) -GalNAc ₃ -1 _(a)682883 GalNAc ₃ -3 _(a-o′)T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds)PS/PO GalNAc₃-3a PO 4899 T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es)^(m)C_(es) ^(m)C_(e) 682884 GalNAc ₃ -7 _(a-o′)T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds)PS/PO GalNAc₃-7a PO 4899 T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es)^(m)C_(es) ^(m)C_(e) 682885 GalNAc ₃ -10 _(a-o′)T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds) PS/POGalNAc₃-10a PO 4899 A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo)^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682886 GalNAc ₃ -13 _(a-o′)T_(es)^(m)C_(eo)T_(eo)T_(eo)Ge_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds) PS/POGalNAc₃-13a PO 4899 A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo)^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 684057 T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds) PS GalNAc₃-19a A_(d) 4900A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(eo) A _(do′) -GalNAc ₃ -19_(a)The legend for Table 85 can be found in Example 74. The structure ofGalNAc₃-1 was shown in Example 9. The structure of GalNAc₃-3_(a) wasshown in Example 39. The structure of GalNAc₃-7_(a) was shown in Example48. The structure of GalNAc₃-10_(a) was shown in Example 46. Thestructure of GalNAc₃-13_(a) was shown in Example 62. The structure ofGalNAc₃-19_(a) was shown in Example 70.

TABLE 84 Antisense inhibition of human TTR in vivo Plasma TTR TTR IsisDosage mRNA protein GalNAc SEQ No. (mg/kg) (% PBS) (% PBS) cluster CM IDNo. PBS n/a 100 100 n/a n/a 420915 6 99 95 n/a n/a 4899 20 48 65 60 1828 660261 0.6 113 87 GalNAc₃-1a A_(d) 4900 2 40 56 6 20 27 20 9 11

TABLE 85 Antisense inhibition of human TTR in vivo TTR Plasma TTRprotein (% PBS at BL) SEQ Dosage mRNA Day 17 GalNAc ID Isis No. (mg/kg)(% PBS) BL Day 3 Day 10 (After sac) cluster CM No. PBS n/a 100 100 96 90114 n/a n/a 420915 6 74 106 86 76 83 n/a n/a 4899 20 43 102 66 61 58 6024 92 43 29 32 682883 0.6 60 88 73 63 68 GalNAc₃- PO 4899 2 18 75 38 2323 3a 6 10 80 35 11 9 682884 0.6 56 88 78 63 67 GalNAc₃- PO 4899 2 19 7644 25 23 7a 6 15 82 35 21 24 682885 0.6 60 92 77 68 76 GalNAc₃- PO 48992 22 93 58 32 32 10a 6 17 85 37 25 20 682886 0.6 57 91 70 64 69 GalNAc₃-PO 4899 2 21 89 50 31 30 13a 6 18 102 41 24 27 684057 0.6 53 80 69 56 62GalNAc₃- A_(d) 4900 2 21 92 55 34 30 19a 6 11 82 50 18 13

TABLE 86 Transaminase levels, body weight changes, and relative organweights ALT (U/L) AST (U/L) Body Liver Spleen Kidney SEQ Dosage Day DayDay Day (% (% (% (% ID Isis No. (mg/kg) BL Day 3 10 17 BL Day 3 10 17BL) PBS) PBS) PBS) No. PBS n/a 33 34 33 24 58 62 67 52 105 100 100 100n/a 420915 6 34 33 27 21 64 59 73 47 115 99 89 91 4899 20 34 30 28 19 6454 56 42 111 97 83 89 60 34 35 31 24 61 58 71 58 113 102 98 95 6602610.6 33 38 28 26 70 71 63 59 111 96 99 92 4900 2 29 32 31 34 61 60 68 61118 100 92 90 6 29 29 28 34 58 59 70 90 114 99 97 95 20 33 32 28 33 6454 68 95 114 101 106 92

TABLE 87 Transaminase levels, body weight changes, and relative organweights ALT (U/L) AST (U/L) Body Liver Spleen Kidney SEQ Dosage Day DayDay Day (% (% (% (% ID Isis No. (mg/kg) BL Day 3 10 17 BL Day 3 10 17BL) PBS) PBS) PBS) No. PBS n/a 32 34 37 41 62 78 76 77 104 100 100 100n/a 420915 6 32 30 34 34 61 71 72 66 102 103 102 105 4899 20 41 34 37 3380 76 63 54 106 107 135 101 60 36 30 32 34 58 81 57 60 106 105 104 99682883 0.6 32 35 38 40 53 81 74 76 104 101 112 95 4899 2 38 39 42 43 7184 70 77 107 98 116 99 6 35 35 41 38 62 79 103 65 105 103 143 97 6828840.6 33 32 35 34 70 74 75 67 101 100 130 99 4899 2 31 32 38 38 63 77 6655 104 103 122 100 6 38 32 36 34 65 85 80 62 99 105 129 95 682885 0.6 3926 37 35 63 63 77 59 100 109 109 112 4899 2 30 26 38 40 54 56 71 72 10298 111 102 6 27 27 34 35 46 52 56 64 102 98 113 96 682886 0.6 30 40 3436 58 87 54 61 104 99 120 101 4899 2 27 26 34 36 51 55 55 69 103 91 10592 6 40 28 34 37 107 54 61 69 109 100 102 99 684057 0.6 35 26 33 39 5651 51 69 104 99 110 102 4900 2 33 32 31 40 54 57 56 87 103 100 112 97 639 33 35 40 67 52 55 92 98 104 121 108

Example 87: Duration of Action In Vivo by Single Doses ofOligonucleotides Targeting TTR Comprising a GalNAc₃ Cluster

ISIS numbers 420915 and 660261 (see Table 83) were tested in a singledose study for duration of action in mice. ISIS numbers 420915, 682883,and 682885 (see Table 83) were also tested in a single dose study forduration of action in mice.

Treatment

Eight week old, male transgenic mice that express human TTR were eachinjected subcutaneously once with 100 mg/kg ISIS No. 420915 or 13.5mg/kg ISIS No. 660261. Each treatment group consisted of 4 animals. Tailbleeds were performed before dosing to determine baseline and at days 3,7, 10, 17, 24, and 39 following the dose. Plasma TTR protein levels weremeasured as described in Example 86. The results below are presented asthe average percent of plasma TTR levels for each treatment group,normalized to baseline levels.

TABLE 88 Plasma TTR protein levels ISIS Dosage Time point GalNAc₃ No.(mg/kg) (days post-dose) TTR (% baseline) Cluster CM SEQ ID No. 420915100 3 30 n/a n/a 4899 7 23 10 35 17 53 24 75 39 100 660261 13.5 3 27GalNAc₃-1a A_(d) 4900 7 21 10 22 17 36 24 48 39 69

Treatment

Female transgenic mice that express human TTR were each injectedsubcutaneously once with 100 mg/kg ISIS No. 420915, 10.0 mg/kg ISIS No.682883, or 10.0 mg/kg 682885. Each treatment group consisted of 4animals. Tail bleeds were performed before dosing to determine baselineand at days 3, 7, 10, 17, 24, and 39 following the dose. Plasma TTRprotein levels were measured as described in Example 86. The resultsbelow are presented as the average percent of plasma TTR levels for eachtreatment group, normalized to baseline levels.

TABLE 89 Plasma TTR protein levels ISIS Dosage Time point GalNAc₃ No.(mg/kg) (days post-dose) TTR (% baseline) Cluster CM SEQ ID No. 420915100 3 48 n/a n/a 4899 7 48 10 48 17 66 31 80 682883 10.0 3 45 GalNAc₃-3aPO 4899 7 37 10 38 17 42 31 65 682885 10.0 3 40 GalNAc₃-10a PO 4899 7 3310 34 17 40 31 64The results in Tables 88 and 89 show that the oligonucleotidescomprising a GalNAc conjugate are more potent with a longer duration ofaction than the parent oligonucleotide lacking a conjugate (ISIS420915).

Example 88: Splicing Modulation In Vivo by Oligonucleotides TargetingSMN Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 90 were tested for splicingmodulation of human survival of motor neuron (SMN) in mice.

TABLE 90 Modified ASOs targeting SMN ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 387954 A_(es)T_(es)T_(es) ^(m)C_(es)A_(es)^(m)C_(es)T_(es)T_(es)T_(es)^(m)C_(es)A_(es)T_(es)A_(es)A_(es)T_(es)G_(es) ^(m)C_(es)T_(es)G_(es)n/a n/a 4901 G_(e) 699819 GalNAc ₃ -7 _(a)-_(o′)A_(es)T_(es)T_(es)^(m)C_(es)A_(es) ^(m)C_(es)T_(es)T_(es)T_(es)^(m)C_(es)A_(es)T_(es)A_(es)A_(es) GalNAc₃-7a PO 4901 T_(es)G_(es)^(m)C_(es)T_(es)G_(es)G_(e) 699821 GalNAc ₃ -7_(a)-_(o′)A_(es)T_(eo)T_(eo) ^(m)C_(eo)A_(eo)^(m)C_(eo)T_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(eo)T_(eo)A_(eo) GalNAc₃-7a PO4901 A_(eo)T_(eo)G_(eo) ^(m)C_(eo)T_(es)G_(es)G_(e) 700000A_(es)T_(es)T_(es) ^(m)C_(es)A_(es) ^(m)C_(es)T_(es)T_(es)T_(es)^(m)C_(es)A_(es)T_(es)A_(es)A_(es)T_(es)G_(es) ^(m)C_(es)T_(es)G_(es)GalNAc₃-1a A_(d) 4902 G_(eo) A _(do′) -GalNAc ₃ -1 _(a) 703421X-ATT^(m)CA^(m)CTTT^(m)CATAATG^(m)CTGG n/a n/a 4901 703422 GalNAc ₃ -7_(b)-X-ATT^(m)CA^(m)CTTT^(m)CATAATG^(m)CTGG GalNAc₃-7b n/a 4901The structure of GalNAc₃-7_(a) was shown previously in Example 48. “X”indicates a 5′ primary amine generated by Gene Tools (Philomath, Oreg.),and GalNAc₃-7_(b) indicates the structure of GalNAc₃-7_(a) lacking the—NH—C₆—O portion of the linker as shown below:

ISIS numbers 703421 and 703422 are morphlino oligonucleotides, whereineach nucleotide of the two oligonucleotides is a morpholino nucleotide.

Treatment

Six week old transgenic mice that express human SMN were injectedsubcutaneously once with an oligonucleotide listed in Table 91 or withsaline. Each treatment group consisted of 2 males and 2 females. Themice were sacrificed 3 days following the dose to determine the liverhuman SMN mRNA levels both with and without exon 7 using real-time PCRaccording to standard protocols. Total RNA was measured using Ribogreenreagent. The SMN mRNA levels were normalized to total mRNA, and furthernormalized to the averages for the saline treatment group. The resultingaverage ratios of SMN mRNA including exon 7 to SMN mRNA missing exon 7are shown in Table 91. The results show that fully modifiedoligonucleotides that modulate splicing and comprise a GalNAc conjugateare significantly more potent in altering splicing in the liver than theparent oligonucleotides lacking a GlaNAc conjugate. Furthermore, thistrend is maintained for multiple modification chemistries, including2′-MOE and morpholino modified oligonucleotides.

TABLE 91 Effect of oligonucleotides targeting human SMN in vivo DoseGalNAc₃ SEQ ISIS No. (mg/kg) +Exon 7/−Exon 7 Cluster CM ID No. Salinen/a 1.00 n/a n/a n/a 387954 32 1.65 n/a n/a 4901 387954 288 5.00 n/a n/a4901 699819 32 7.84 GalNAc₃-7a PO 4901 699821 32 7.22 GalNAc₃-7a PO 4901700000 32 6.91 GalNAc₃-1a A_(d) 4902 703421 32 1.27 n/a n/a 4901 70342232 4.12 GalNAc₃-7b n/a 4901

Example 89: Antisense Inhibition In Vivo by Oligonucleotides TargetingApolipoprotein A (Apo(a)) Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 92 below were tested in a study fordose-dependent inhibition of Apo(a) in transgenic mice.

TABLE 92 Modified ASOs targeting Apo(a) ISIS GalNAc₃ SEQ ID No.Sequences (5′ to 3′) Cluster CM No. 494372 T_(es)G_(es) ^(m)C_(es)T_(es)^(m)C_(es) ^(m)C_(ds) n/a n/a 4903 G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(es)G_(es)T_(es) T_(es) ^(m)C_(e) 681257 GalNAc₃ -7 _(a)-_(o′)T_(es)G_(eo) GalNAc₃-7a PO 4903 ^(m)C_(eo)T_(eo)^(m)C_(eo) ^(m)C_(ds)G_(ds)T_(ds) T_(ds)G_(ds)G_(ds)T_(ds)G_(ds)^(m)C_(ds) T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e)The structure of GalNAc₃-7a was shown in Example 48.

Treatment

Eight week old, female C57BL/6 mice (Jackson Laboratory, Bar Harbor,Me.) were each injected subcutaneously once per week at a dosage shownbelow, for a total of six doses, with an oligonucleotide listed in Table92 or with PBS. Each treatment group consisted of 3-4 animals. Tailbleeds were performed the day before the first dose and weekly followingeach dose to determine plasma Apo(a) protein levels. The mice weresacrificed two days following the final administration. Apo(a) livermRNA levels were determined using real-time PCR and RIBOGREEN RNAquantification reagent (Molecular Probes, Inc. Eugene, Oreg.) accordingto standard protocols. Apo(a) plasma protein levels were determinedusing ELISA, and liver transaminase levels were determined. The mRNA andplasma protein results in Table 93 are presented as the treatment groupaverage percent relative to the PBS treated group. Plasma protein levelswere further normalized to the baseline (BL) value for the PBS group.Average absolute transaminase levels and body weights (% relative tobaseline averages) are reported in Table 94.

As illustrated in Table 93, treatment with the oligonucleotides loweredApo(a) liver mRNA and plasma protein levels in a dose-dependent manner.Furthermore, the oligonucleotide comprising the GalNAc conjugate wassignificantly more potent with a longer duration of action than theparent oligonucleotide lacking a GalNAc conjugate. As illustrated inTable 94, transaminase levels and body weights were unaffected by theoligonucleotides, indicating that the oligonucleotides were welltolerated.

TABLE 93 Apo(a) liver mRNA and plasma protein levels ISIS Dosage Apo(a)mRNA Apo(a) plasma protein (% PBS) No. (mg/kg) (% PBS) BL Week 1 Week 2Week 3 Week 4 Week 5 Week 6 PBS n/a 100 100 120 119 113 88 121 97 4943723 80 84 89 91 98 87 87 79 10 30 87 72 76 71 57 59 46 30 5 92 54 28 10 79 7 681257 0.3 75 79 76 89 98 71 94 78 1 19 79 88 66 60 54 32 24 3 2 8252 17 7 4 6 5 10 2 79 17 6 3 2 4 5

TABLE 94 Dosage ALT AST Body weight ISIS No. (mg/kg) (U/L) (U/L) (%baseline) PBS n/a 37 54 103 494372 3 28 68 106 10 22 55 102 30 19 48 103681257 0.3 30 80 104 1 26 47 105 3 29 62 102 10 21 52 107

Example 90: Antisense Inhibition In Vivo by Oligonucleotides TargetingTTR Comprising a GalNAc₃ Cluster

Oligonucleotides listed in Table 95 below were tested in adose-dependent study for antisense inhibition of human transthyretin(TTR) in transgenic mice that express the human TTR gene.

Treatment

TTR transgenic mice were each injected subcutaneously once per week forthree weeks, for a total of three doses, with an oligonucleotide anddosage listed in Table 96 or with PBS. Each treatment group consisted of4 animals. Prior to the first dose, a tail bleed was performed todetermine plasma TTR protein levels at baseline (BL). The mice weresacrificed 72 hours following the final administration. TTR proteinlevels were measured using a clinical analyzer (AU480, Beckman Coulter,CA). Real-time PCR and RIBOGREEN RNA quantification reagent (MolecularProbes, Inc. Eugene, Oreg.) were used according to standard protocols todetermine liver human TTR mRNA levels. The results presented in Table 96are the average values for each treatment group. The mRNA levels are theaverage values relative to the average for the PBS group. Plasma proteinlevels are the average values relative to the average value for the PBSgroup at baseline. “BL” indicates baseline, measurements that were takenjust prior to the first dose. As illustrated in Table 96, treatment withantisense oligonucleotides lowered TTR expression levels in adose-dependent manner. The oligonucleotides comprising a GalNAcconjugate were more potent than the parent lacking a GalNAc conjugate(ISIS 420915), and oligonucleotides comprising a phosphodiester ordeoxyadenosine cleavable moiety showed significant improvements inpotency compared to the parent lacking a conjugate (see ISIS numbers682883 and 666943 vs 420915 and see Examples 86 and 87).

TABLE 95 Oligonucleotides targeting human TTR GalNAc SEQ Isis No.Sequence 5′ to 3′ Linkages cluster CM ID No. 420915 T_(es)^(m)C_(es)T_(es)T_(es)G_(es)G_(ds)T_(ds)T_(ds)A_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds) PS n/a n/a 4899 A_(es)T_(es)^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682883 GalNAc ₃ -3 _(a-o′)T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds)PS/PO GalNAc₃-3a PO 4899 T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es)^(m)C_(es) ^(m)C_(e) 666943 GalNAc ₃ -3 _(a-o′) A _(do)T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) PS/PO GalNAc₃-3aA_(d) 4904 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo)^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682887 GalNAc ₃ -7 _(a-o′) A _(do)T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) PS/PO GalNAc₃-7aA_(d) 4904 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo)^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682888 GalNAc ₃ -10 _(a-o′) A_(do)T_(es) ^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) PS/POGalNAc₃-10a A_(d) 4904^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es)^(m)C_(es) ^(m)C_(e) 682889 GalNAc ₃ -13 _(a-o′) A _(do)T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) PS/PO GalNAc₃-13aA_(d) 4904 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo)^(m)C_(es) ^(m)C_(es) ^(m)C_(e)The legend for Table 95 can be found in Example 74. The structure ofGalNAc₃-3_(a) was shown in Example 39. The structure of GalNAc₃-7_(a)was shown in Example 48. The structure of GalNAc₃-10_(a) was shown inExample 46. The structure of GalNAc₃-13_(a) was shown in Example 62.

TABLE 96 Antisense inhibition of human TTR in vivo Dosage TTR mRNA TTRprotein GalNAc Isis No. (mg/kg) (% PBS) (% BL) cluster CM PBS n/a 100124 n/a n/a 420915 6 69 114 n/a n/a 20 71 86 60 21 36 682883 0.6 61 73GalNAc₃-3a PO 2 23 36 6 18 23 666943 0.6 74 93 GalNAc₃-3a A_(d) 2 33 576 17 22 682887 0.6 60 97 GalNAc₃-7a A_(d) 2 36 49 6 12 19 682888 0.6 6592 GalNAc₃-10a A_(d) 2 32 46 6 17 22 682889 0.6 72 74 GalNAc₃-13a A_(d)2 38 45 6 16 18

Example 91: Antisense Inhibition In Vivo by Oligonucleotides TargetingFactor VII Comprising a GalNAc₃ Conjugate in Non-Human Primates

Oligonucleotides listed in Table 97 below were tested in a non-terminal,dose escalation study for antisense inhibition of Factor VII in monkeys.

Treatment

Non-naïve monkeys were each injected subcutaneously on days 0, 15, and29 with escalating doses of an oligonucleotide listed in Table 97 orwith PBS. Each treatment group consisted of 4 males and 1 female. Priorto the first dose and at various time points thereafter, blood drawswere performed to determine plasma Factor VII protein levels. Factor VIIprotein levels were measured by ELISA. The results presented in Table 98are the average values for each treatment group relative to the averagevalue for the PBS group at baseline (BL), the measurements taken justprior to the first dose. As illustrated in Table 98, treatment withantisense oligonucleotides lowered Factor VII expression levels in adose-dependent manner, and the oligonucleotide comprising the GalNAcconjugate was significantly more potent in monkeys compared to theoligonucleotide lacking a GalNAc conjugate.

TABLE 97 Oligonucleotides targeting Factor VII GalNAc SEQ Isis No.Sequence 5′ to 3′ Linkages cluster CM ID No. 407935 A_(es)T_(es)G_(es)^(m)C_(es)A_(es)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds)A_(ds)T_(ds)G_(ds)^(m)C_(ds)T_(ds) PS n/a n/a 4905 T_(es) ^(m)C_(es)T_(es)G_(es)A_(e)686892 GalNAc ₃ -10 _(a-o′)A_(es)T_(es)G_(es)^(m)C_(es)A_(es)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) PS GalNAc₃-10a PO 4905A_(ds)T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)T_(es)G_(es)A_(e)The legend for Table 97 can be found in Example 74. The structure ofGalNAc₃-10_(a) was shown in Example 46.

TABLE 98 Factor VII plasma protein levels Dose Factor VII ISIS No. Day(mg/kg) (% BL) 407935 0 n/a 100 15 10 87 22 n/a 92 29 30 77 36 n/a 46 43n/a 43 686892 0  3 100 15 10 56 22 n/a 29 29 30 19 36 n/a 15 43 n/a 11

Example 92: Antisense Inhibition in Primary Hepatocytes by AntisenseOligonucleotides Targeting ApoCIII Comprising a GalNAc₃ Conjugate

Primary mouse hepatocytes were seeded in 96-well plates at 15,000 cellsper well, and the oligonucleotides listed in Table 99, targeting mouseApoC-III, were added at 0.46, 1.37, 4.12, or 12.35, 37.04, 111.11, or333.33 nM or 1.00 μM. After incubation with the oligonucleotides for 24hours, the cells were lysed and total RNA was purified using RNeasy(Qiagen). ApoC-III mRNA levels were determined using real-time PCR andRIBOGREEN® RNA quantification reagent (Molecular Probes, Inc.) accordingto standard protocols. IC₅₀ values were determined using Prism 4software (GraphPad). The results show that regardless of whether thecleavable moiety was a phosphodiester or a phosphodiester-linkeddeoxyadensoine, the oligonucleotides comprising a GalNAc conjugate weresignificantly more potent than the parent oligonucleotide lacking aconjugate.

TABLE 99 Inhibition of mouse APOC-III expression in mouse primaryhepatocytes ISIS IC₅₀ SEQ No. Sequence (5′ to 3′) CM (nM) ID No. 440670^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)^(m)C_(es)A_(es)G_(es) ^(m)C_(es)A_(e) n/a 13.20 4906 661180^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)^(m)C_(es) A_(d) 1.40 4907 A_(es)G_(es) ^(m)C_(es)A_(eo) A _(do′)-GalNAc ₃ -1 _(a) 680771 GalNAc ₃ -3 _(a-o′) ^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)^(m)C_(es) PO 0.70 4906 A_(es)G_(es) ^(m)C_(es)A_(e) 680772 GalNAc ₃ -7_(a-o′) ^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)^(m)C_(es) PO 1.70 4906 A_(es)G_(es) ^(m)C_(es)A_(e) 680773 GalNAc ₃ -10_(a-o′) ^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)^(m)C_(es) PO 2.00 4906 A_(es)G_(es) ^(m)C_(es)A_(e) 680774 GalNAc ₃ -13_(a-o′) ^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)^(m)C_(es) PO 1.50 4906 A_(es)G_(es) ^(m)C_(es)A_(e) 681272 GalNAc ₃ -3_(a-o′) ^(m)C_(es)A_(eo)G_(eo)^(m)C_(eo)T_(eo)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)^(m)C_(eo) PO <0.46 4906 A_(eo)G_(es) ^(m)C_(es)A_(e) 681273 GalNAc ₃ -3_(a)-_(o′) A _(do) ^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)A_(d) 1.10 4908 ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)A_(e) 683733^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)^(m)C_(es) A_(d) 2.50 4907 A_(es)G_(es) ^(m)C_(es)A_(eo) A _(do′)-GalNAc ₃ -19 _(a)The structure of GalNAc₃-1_(a) was shown previously in Example 9,GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-7_(a) was shown inExample 48, GalNAc₃-10_(a) was shown in Example 46, GalNAc₃-13_(a) wasshown in Example 62, and GalNAc₃-19_(a) was shown in Example 70.

Example 93: Antisense Inhibition In Vivo by Oligonucleotides TargetingSRB-1 Comprising Mixed Wings and a 5′-GalNAc₃ Conjugate

The oligonucleotides listed in Table 100 were tested in a dose-dependentstudy for antisense inhibition of SRB-1 in mice.

TABLE 100 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 449093 T_(ks)T_(ks)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(ks) ^(m)C_(k) n/a n/a 4909 699806 GalNAc ₃-3 _(a)-_(o′)T_(ks)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-3a PO 4909T_(ds)T_(ks) ^(m)C_(ks) ^(m)C_(k) 699807 GalNAc ₃ -7_(a)-_(o′)T_(ks)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-7a PO 4909T_(ds)T_(ks) ^(m)C_(ks) ^(m)C_(k) 699809 GalNAc ₃ -7_(a)-_(o′)T_(ks)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-7a PO 4909T_(ds)T_(es) ^(m)C_(es) ^(m)C_(e) 699811 GalNAc ₃ -7_(a)-_(o′)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-7a PO 4909T_(ds)T_(ks) ^(m)C_(ks) ^(m)C_(k) 699813 GalNAc ₃ -7_(a)-_(o′)T_(ks)T_(ds) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-7a PO 4909T_(ds)T_(ks) ^(m)C_(ds) ^(m)C_(k) 699815 GalNAc ₃ -7_(a)-_(o′)T_(es)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-7a PO 4909T_(ds)T_(ks) ^(m)C_(ks) ^(m)C_(e)

The structure of GalNAc₃-3_(a) was shown previously in Example 39, andthe structure of GalNAc₃-7a was shown previously in Example 48.Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicatesβ-D-2′-deoxyribonucleoside; “k” indicates 6′-(S)—CH₃ bicyclic nucleoside(cEt); “s” indicates phosphorothioate internucleoside linkages (PS); “o”indicates phosphodiester internucleoside linkages (PO). Superscript “m”indicates 5-methylcytosines.

Treatment

Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.)were injected subcutaneously once at the dosage shown below with anoligonucleotide listed in Table 100 or with saline. Each treatment groupconsisted of 4 animals. The mice were sacrificed 72 hours following thefinal administration. Liver SRB-1 mRNA levels were measured usingreal-time PCR. SRB-1 mRNA levels were normalized to cyclophilin mRNAlevels according to standard protocols. The results are presented as theaverage percent of SRB-1 mRNA levels for each treatment group relativeto the saline control group. As illustrated in Table 101, treatment withantisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependentmanner, and the gapmer oligonucleotides comprising a GalNAc conjugateand having wings that were either full cEt or mixed sugar modificationswere significantly more potent than the parent oligonucleotide lacking aconjugate and comprising full cEt modified wings.

Body weights, liver transaminases, total bilirubin, and BUN were alsomeasured, and the average values for each treatment group are shown inTable 101. Body weight is shown as the average percent body weightrelative to the baseline body weight (% BL) measured just prior to theoligonucleotide dose.

TABLE 101 SRB-1 mRNA, ALT, AST, BUN, and total bilirubin levels and bodyweights Body ISIS Dosage SRB-1 mRNA ALT AST weight No. (mg/kg) (% PBS)(U/L) (U/L) Bil BUN (% BL) PBS n/a 100 31 84 0.15 28 102 449093 1 111 1848 0.17 31 104 3 94 20 43 0.15 26 103 10 36 19 50 0.12 29 104 699806 0.1114 23 58 0.13 26 107 0.3 59 21 45 0.12 27 108 1 25 30 61 0.12 30 104699807 0.1 121 19 41 0.14 25 100 0.3 73 23 56 0.13 26 105 1 24 22 690.14 25 102 699809 0.1 125 23 57 0.14 26 104 0.3 70 20 49 0.10 25 105 133 34 62 0.17 25 107 699811 0.1 123 48 77 0.14 24 106 0.3 94 20 45 0.1325 101 1 66 57 104 0.14 24 107 699813 0.1 95 20 58 0.13 28 104 0.3 98 2261 0.17 28 105 1 49 19 47 0.11 27 106 699815 0.1 93 30 79 0.17 25 1050.3 64 30 61 0.12 26 105 1 24 18 41 0.14 25 106

Example 94: Antisense Inhibition In Vivo by Oligonucleotides TargetingSRB-1 Comprising 2′-Sugar Modifications and a 5′-GalNAc₃ Conjugate

The oligonucleotides listed in Table 102 were tested in a dose-dependentstudy for antisense inhibition of SRB-1 in mice.

TABLE 102 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 353382 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es) n/a n/a 4886 T_(es)T_(e)700989 G_(ms)C_(ms)U_(ms)U_(ms)C_(ms)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)U_(ms)C_(ms)C_(ms)n/a n/a 4910 U_(ms)U_(m) 666904 GalNAc ₃ -3 _(a) - _(o′)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) GalNAc₃-3a PO 4886^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 700991 GalNAc ₃-7 _(a) - _(o′)G_(ms)C_(ms)U_(ms)U_(ms)C_(ms)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds) GalNAc₃-7a PO 4910 A_(ds)^(m)C_(ds)T_(ds)U_(ms)C_(ms)C_(ms)U_(ms)U_(m)Subscript “m” indicates a 2′-O-methyl modified nucleoside. See Example74 for complete table legend. The structure of GalNAc₃-3_(a) was shownpreviously in Example 39, and the structure of GalNAc₃-7a was shownpreviously in Example 48.

Treatment

The study was completed using the protocol described in Example 93.Results are shown in Table 103 below and show that both the 2′-MOE and2′-OMe modified oligonucleotides comprising a GalNAc conjugate weresignificantly more potent than the respective parent oligonucleotideslacking a conjugate. The results of the body weights, livertransaminases, total bilirubin, and BUN measurements indicated that thecompounds were all well tolerated.

TABLE 103 SRB-1 mRNA Dosage SRB-1 mRNA ISIS No. (mg/kg) (% PBS) PBS n/a100 353382 5 116 15 58 45 27 700989 5 120 15 92 45 46 666904 1 98 3 4510 17 700991 1 118 3 63 10 14

Example 95: Antisense Inhibition In Vivo by Oligonucleotides TargetingSRB-1 Comprising Bicyclic Nucleosides and a 5′-GalNAc₃ Conjugate

The oligonucleotides listed in Table 104 were tested in a dose-dependentstudy for antisense inhibition of SRB-1 in mice.

TABLE 104 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No 440762 T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) n/an/a 4880 666905 GalNAc ₃ -3 _(a) - _(o′)T_(ks)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds) A_(ds)^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) GalNAc₃-3_(a) PO 4880 699782 GalNAc ₃-7 _(a) - _(o′)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k)GalNAc₃-7_(a) PO 4880 699783 GalNAc ₃ -3 _(a) - _(o′)T_(ls)^(m)C_(ls)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(ls) ^(m)C_(l) GalNAc₃-3_(a) PO 4880 653621 T_(ls)^(m)C_(ls)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(ls) ^(m)C_(lo) A _(do′) -GalNAc ₃ -1 _(a)GalNAc₃-1_(a) A_(d) 4881 439879 T_(gs) ^(m)C_(gs)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(d)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(gs) ^(m)C_(g) n/an/a 4880 699789 GalNAc ₃ -3 _(a) - _(o′)T_(gs)^(m)C_(gs)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(d)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(gs) ^(m)C_(g) GalNAc₃-3_(a) PO 4880Subscript “g” indicates a fluoro-HNA nucleoside, subscript “1” indicatesa locked nucleoside comprising a 2′-O—CH₂-4′ bridge. See the Example 74table legend for other abbreviations. The structure of GalNAc₃-1_(a) wasshown previously in Example 9, the structure of GalNAc₃-3_(a) was shownpreviously in Example 39, and the structure of GalNAc₃-7a was shownpreviously in Example 48.

Treatment

The study was completed using the protocol described in Example 93.Results are shown in Table 105 below and show that oligonucleotidescomprising a GalNAc conjugate and various bicyclic nucleosidemodifications were significantly more potent than the parentoligonucleotide lacking a conjugate and comprising bicyclic nucleosidemodifications. Furthermore, the oligonucleotide comprising a GalNAcconjugate and fluoro-HNA modifications was significantly more potentthan the parent lacking a conjugate and comprising fluoro-HNAmodifications. The results of the body weights, liver transaminases,total bilirubin, and BUN measurements indicated that the compounds wereall well tolerated.

TABLE 105 SRB-1 mRNA, ALT, AST, BUN, and total bilirubin levels and bodyweights Dosage SRB-1 mRNA ISIS No. (mg/kg) (% PBS) PBS n/a 100 440762 1104 3 65 10 35 666905 0.1 105 0.3 56 1 18 699782 0.1 93 0.3 63 1 15699783 0.1 105 0.3 53 1 12 653621 0.1 109 0.3 82 1 27 439879 1 96 3 7710 37 699789 0.1 82 0.3 69 1 26

Example 96: Plasma Protein Binding of Antisense OligonucleotidesComprising a GalNAc₃ Conjugate Group

Oligonucleotides listed in Table 70 targeting ApoC-III andoligonucleotides in Table 106 targeting Apo(a) were tested in anultra-filtration assay in order to assess plasma protein binding.

TABLE 106 Modified oligonucleotides targeting Apo(a) ISIS GalNAc₃ SEQNo. Sequences (5′ to 3′) Cluster CM ID No 494372 T_(es)G_(es)^(m)C_(es)T_(es) ^(m)C_(es)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(es)G_(es)T_(es) n/a n/a 4903 T_(es) ^(m)C_(e) 693401T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es) n/a n/a 4903 T_(es) ^(m)C_(e) 681251GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(es) ^(m)C_(es)T_(es) ^(m)C_(es)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(ds)GalNAc₃-7_(a) PO 4903 T_(ds)T_(es)G_(es)T_(es)T_(es) ^(m)C_(e) 681257GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(ds)GalNAc₃-7_(a) PO 4903 T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e)See the Example 74 for table legend. The structure of GalNAc₃-7a wasshown previously in Example 48.

Ultrafree-MC ultrafiltration units (30,000 NMWL, low-binding regeneratedcellulose membrane, Millipore, Bedford, Mass.) were pre-conditioned with300 μL of 0.5% Tween 80 and centrifuged at 2000 g for 10 minutes, thenwith 300 μL of a 300 μg/mL solution of a control oligonucleotide in H₂Oand centrifuged at 2000 g for 16 minutes. In order to assessnon-specific binding to the filters of each test oligonucleotide fromTables 70 and 106 to be used in the studies, 300 μL of a 250 ng/mLsolution of oligonucleotide in H₂O at pH 7.4 was placed in thepre-conditioned filters and centrifuged at 2000 g for 16 minutes. Theunfiltered and filtered samples were analyzed by an ELISA assay todetermine the oligonucleotide concentrations. Three replicates were usedto obtain an average concentration for each sample. The averageconcentration of the filtered sample relative to the unfiltered sampleis used to determine the percent of oligonucleotide that is recoveredthrough the filter in the absence of plasma (% recovery).

Frozen whole plasma samples collected in K3-EDTA from normal, drug-freehuman volunteers, cynomolgus monkeys, and CD-1 mice, were purchased fromBioreclamation LLC (Westbury, N.Y.). The test oligonucleotides wereadded to 1.2 mL aliquots of plasma at two concentrations (5 and 150μg/mL). An aliquot (300 μL) of each spiked plasma sample was placed in apre-conditioned filter unit and incubated at 37° C. for 30 minutes,immediately followed by centrifugation at 2000 g for 16 minutes.Aliquots of filtered and unfiltered spiked plasma samples were analyzedby an ELISA to determine the oligonucleotide concentration in eachsample. Three replicates per concentration were used to determine theaverage percentage of bound and unbound oligonucleotide in each sample.The average concentration of the filtered sample relative to theconcentration of the unfiltered sample is used to determine the percentof oligonucleotide in the plasma that is not bound to plasma proteins (%unbound). The final unbound oligonucleotide values are corrected fornon-specific binding by dividing the % unbound by the % recovery foreach oligonucleotide. The final % bound oligonucleotide values aredetermined by subtracting the final % unbound values from 100. Theresults are shown in Table 107 for the two concentrations ofoligonucleotide tested (5 and 150 μg/mL) in each species of plasma. Theresults show that GalNAc conjugate groups do not have a significantimpact on plasma protein binding. Furthermore, oligonucleotides withfull PS internucleoside linkages and mixed PO/PS linkages both bindplasma proteins, and those with full PS linkages bind plasma proteins toa somewhat greater extent than those with mixed PO/PS linkages.

TABLE 107 Percent of modified oligonucleotide bound to plasma proteinsISIS Human plasma Monkey plasma Mouse plasma No. 5 μg/mL 150 μg/mL 5μg/mL 150 μg/mL 5 μg/mL 150 μg/mL 304801 99.2 98.0 99.8 99.5 98.1 97.2663083 97.8 90.9 99.3 99.3 96.5 93.0 674450 96.2 97.0 98.6 94.4 94.689.3 494372 94.1 89.3 98.9 97.5 97.2 93.6 693401 93.6 89.9 96.7 92.094.6 90.2 681251 95.4 93.9 99.1 98.2 97.8 96.1 681257 93.4 90.5 97.693.7 95.6 92.7

Example 97: Modified Oligonucleotides Targeting TTR Comprising a GalNAc₃Conjugate Group

The oligonucleotides shown in Table 108 comprising a GalNAc conjugatewere designed to target TTR.

TABLE 108 Modified oligonucleotides targeting TTR GalNAc₃ SEQ ID ISISNo. Sequences (5′ to 3′) Cluster CM No 666941 GalNAc ₃ -3 _(a) - _(o′) A_(do) T_(es) ^(m) C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-3 A_(d) 4904 A_(ds) T_(ds) G_(ds) A_(ds)A_(ds) A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 666942 T_(es) ^(m)C_(eo) T_(eo) T_(eo) G_(eo) G_(ds) T_(ds) T_(ds) A_(ds) ^(m)C_(ds)A_(ds) T_(ds) G_(ds) A_(ds) A_(ds) GalNAc₃-1 A_(d) 4904 A_(eo) T_(eo)^(m)C_(es) ^(m)C_(es) ^(m)C_(eo) A _(do′) -GalNAc ₃ -3 _(a) 682876GalNAc ₃ -3 _(a) - _(o′)T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds)T_(ds) T_(ds) A_(ds) ^(m)C_(ds) A_(ds) T_(ds) GalNAc₃-3 PO 4899 G_(ds)A_(ds) A_(ds) A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682877GalNAc ₃ -7 _(a) - _(o′)T_(es) ^(m) C_(es) T_(es) T_(es) G_(es) G_(ds)T_(ds) T_(ds) A_(ds) ^(m)C_(ds) A_(ds) T_(ds) GalNAc₃-7 PO 4899 G_(ds)A_(ds) A_(ds) A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682878GalNAc ₃ -10 _(a) - _(o′)T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds)T_(ds) T_(ds) A_(ds) ^(m)C_(ds) A_(ds) GalNAc₃-10 PO 4899 T_(ds) G_(ds)A_(ds) A_(ds) A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682879GalNAc ₃ -13 _(a) - _(o′)T_(es) ^(m)C_(es) T_(es) T_(es) G_(es) G_(ds)T_(ds) T_(ds) A_(ds) ^(m)C_(ds) A_(ds) GalNAc₃-13 PO 4899 T_(ds) G_(ds)A_(ds) A_(ds) A_(es) T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682880GalNAc ₃ -7 _(a) - _(o′) A _(do) T_(es) ^(m)C_(es) T_(es) T_(es) G_(es)G_(ds) T_(ds) T_(ds) A_(ds) ^(m)C_(ds) GalNAc₃-7 A_(d) 4904 A_(ds)T_(ds) G_(ds) A_(ds) A_(ds) A_(es) T_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(e) 682881 GalNAc ₃ -10 _(a) - _(o′) A _(do) T_(es) ^(m)C_(es)T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds) A_(ds) ^(m)C_(ds) GalNAc₃-10A_(d) 4904 A_(ds) T_(ds) G_(ds) A_(ds) A_(ds) A_(es) T_(es) ^(m)C_(es)^(m)C_(es) ^(m)C_(e) 682882 GalNAc ₃ -13 _(a) - _(o′) A _(do) T_(es)^(m)C_(es) T_(es) T_(es) G_(es) G_(ds) T_(ds) T_(ds) A_(ds) ^(m)C_(ds)GalNAc₃-13 A_(d) 4904 A_(ds) T_(ds) G_(ds) A_(ds) A_(ds) A_(es) T_(es)^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 684056 T_(es) ^(m) C_(es) T_(es) T_(es)G_(es) G_(ds) T_(ds) T_(ds) A_(ds) ^(m)C_(ds) A_(ds) T_(ds) G_(ds)A_(ds) A_(ds) GalNAc₃-19 A_(d) 4900 A_(es) T_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(eo) A _(do′) -GalNAc ₃ -19 _(a)The legend for Table 108 can be found in Example 74. The structure ofGalNAc₃-1 was shown in Example 9. The structure of GalNAc₃-3_(a) wasshown in Example 39. The structure of GalNAc₃-7_(a) was shown in Example48. The structure of GalNAc₃-10_(a) was shown in Example 46. Thestructure of GalNAc₃-13_(a) was shown in Example 62. The structure ofGalNAc₃-19_(a) was shown in Example 70.

Example 98: Evaluation of Pro-Inflammatory Effects of OligonucleotidesComprising a GalNAc Conjugate in hPMBC Assay

The oligonucleotides listed in Table 109 and were tested forpro-inflammatory effects in an hPMBC assay as described in Examples 23and 24. (See Tables 30, 83, 95, and 108 for descriptions of theoligonucleotides.) ISIS 353512 is a high responder used as a positivecontrol, and the other oligonucleotides are described in Tables 83, 95,and 108. The results shown in Table 109 were obtained using blood fromone volunteer donor. The results show that the oligonucleotidescomprising mixed PO/PS internucleoside linkages produced significantlylower pro-inflammatory responses compared to the same oligonucleotideshaving full PS linkages. Furthermore, the GalNAc conjugate group did nothave a significant effect in this assay.

TABLE 109 GalNAc₃ ISIS No. E_(max)/EC₅₀ cluster Linkages CM 353512 3630n/a PS n/a 420915 802 n/a PS n/a 682881 1311 GalNAc₃-10 PS A_(d) 6828880.26 GalNAc₃-10 PO/PS A_(d) 684057 1.03 GalNAc₃-19 PO/PS A_(d)

Example 99: Binding Affinities of Oligonucleotides Comprising a GalNAcConjugate for the Asialoglycoprotein Receptor

The binding affinities of the oligonucleotides listed in Table 110 (seeTable 76 for descriptions of the oligonucleotides) for theasialoglycoprotein receptor were tested in a competitive receptorbinding assay. The competitor ligand, α1-acid glycoprotein (AGP), wasincubated in 50 mM sodium acetate buffer (pH 5) with 1 Uneuraminidase-agarose for 16 hours at 37° C., and >90% desialylation wasconfirmed by either sialic acid assay or size exclusion chromatography(SEC). Iodine monochloride was used to iodinate the AGP according to theprocedure by Atsma et al. (see J Lipid Res. 1991 January; 32(1):173-81.) In this method, desialylated α1-acid glycoprotein (de-AGP) wasadded to 10 mM iodine chloride, Na¹²⁵I, and 1 μM glycine in 0.25 μMNaOH. After incubation for 10 minutes at room temperature, ¹²⁵I-labeledde-AGP was separated from free ¹²⁵I by concentrating the mixture twiceutilizing a 3 KDMWCO spin column. The protein was tested for labelingefficiency and purity on a HPLC system equipped with an Agilent SEC-3column (7.8×300 mm) and a ß-RAM counter. Competition experimentsutilizing ¹²⁵I-labeled de-AGP and various GalNAc-cluster containing ASOswere performed as follows. Human HepG2 cells (10⁶ cells/ml) were platedon 6-well plates in 2 ml of appropriate growth media. MEM mediasupplemented with 10% fetal bovine serum (FBS), 2 mM L-Glutamine and 10mM HEPES was used. Cells were incubated 16-20 hours @ 37° C. with 5% and10% CO₂ respectively. Cells were washed with media without FBS prior tothe experiment. Cells were incubated for 30 min @37° C. with 1 mlcompetition mix containing appropriate growth media with 2% FBS, 10⁻⁸ μM¹²⁵I-labeled de-AGP and GalNAc-cluster containing ASOs at concentrationsranging from 10⁻¹¹ to 10⁻⁵ μM. Non-specific binding was determined inthe presence of 10⁻² μM GalNAc sugar. Cells were washed twice with mediawithout FBS to remove unbound ¹²⁵I-labeled de-AGP and competitor GalNAcASO. Cells were lysed using Qiagen's RLT buffer containing 1%β-mercaptoethanol. Lysates were transferred to round bottom assay tubesafter a brief 10 min freeze/thaw cycle and assayed on a γ-counter.Non-specific binding was subtracted before dividing ¹²⁵I protein countsby the value of the lowest GalNAc-ASO concentration counts. Theinhibition curves were fitted according to a single site competitionbinding equation using a nonlinear regression algorithm to calculate thebinding affinities (K_(D)'s).

The results in Table 110 were obtained from experiments performed onfive different days. Results for oligonucleotides marked withsuperscript “a” are the average of experiments run on two differentdays. The results show that the oligonucleotides comprising a GalNAcconjugate group on the 5′-end bound the asialoglycoprotein receptor onhuman HepG2 cells with 1.5 to 16-fold greater affinity than theoligonucleotides comprising a GalNAc conjugate group on the 3′-end.

TABLE 110 Asialoglycoprotein receptor binding assay resultsOligonucleotide end to GalNAc which GalNAc K_(D) ISIS No. conjugateconjugate is attached (nM) 661161^(a) GalNAc₃-3 5′ 3.7 666881^(a)GalNAc₃-10 5′ 7.6 666981  GalNAc₃-7 5′ 6.0 670061  GalNAc₃-13 5′ 7.4655861^(a) GalNAc₃-1 3′ 11.6 677841^(a) GalNAc₃-19 3′ 60.8

Example 100: Antisense Inhibition In Vivo by Oligonucleotides Comprisinga GalNAc Conjugate Group Targeting Apo(a) In Vivo

The oligonucleotides listed in Table 111a below were tested in a singledose study for duration of action in mice.

TABLE 111a Modified ASOs targeting APO(a) ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 681251 GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(es)^(m)C_(es)T_(es) ^(m)C_(es) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)GalNAc₃-7a PO 4903 T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(es)G_(es)T_(es)T_(es)^(m)C_(e) 681257 GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo)^(m)C_(eo) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-7a PO 4903T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e)

The structure of GalNAc₃-7_(a) was shown in Example 48.

Treatment

Female transgenic mice that express human Apo(a) were each injectedsubcutaneously once per week, for a total of 6 doses, with anoligonucleotide and dosage listed in Table 111b or with PBS. Eachtreatment group consisted of 3 animals. Blood was drawn the day beforedosing to determine baseline levels of Apo(a) protein in plasma and at72 hours, 1 week, and 2 weeks following the first dose. Additional blooddraws will occur at 3 weeks, 4 weeks, 5 weeks, and 6 weeks following thefirst dose. Plasma Apo(a) protein levels were measured using an ELISA.The results in Table 11 lb are presented as the average percent ofplasma Apo(a) protein levels for each treatment group, normalized tobaseline levels (% BL), The results show that the oligonucleotidescomprising a GalNAc conjugate group exhibited potent reduction in Apo(a)expression. This potent effect was observed for the oligonucleotide thatcomprises full PS internucleoside linkages and the oligonucleotide thatcomprises mixed PO and PS linkages.

TABLE 111b Apo(a) plasma protein levels Apo(a) at Apo(a) at Apo(a) atDosage 72 hours 1 week 3 weeks ISIS No. (mg/kg) (% BL) (% BL) (% BL) PBSn/a 116 104 107 681251 0.3 97 108 93 1.0 85 77 57 3.0 54 49 11 10.0 2315 4 681257 0.3 114 138 104 1.0 91 98 54 3.0 69 40 6 10.0 30 21 4

Example 101: Antisense Inhibition by Oligonucleotides Comprising aGalNAc Cluster Linked Via a Stable Moiety

The oligonucleotides listed in Table 112 were tested for inhibition ofmouse APOC-III expression in vivo. C57Bl/6 mice were each injectedsubcutaneously once with an oligonucleotide listed in Table 112 or withPBS. Each treatment group consisted of 4 animals. Each mouse treatedwith ISIS 440670 received a dose of 2, 6, 20, or 60 mg/kg. Each mousetreated with ISIS 680772 or 696847 received 0.6, 2, 6, or 20 mg/kg. TheGalNAc conjugate group of ISIS 696847 is linked via a stable moiety, aphosphorothioate linkage instead of a readily cleavable phosphodiestercontaining linkage. The animals were sacrificed 72 hours after the dose.Liver APOC-III mRNA levels were measured using real-time PCR. APOC-IIImRNA levels were normalized to cyclophilin mRNA levels according tostandard protocols. The results are presented in Table 112 as theaverage percent of APOC-III mRNA levels for each treatment grouprelative to the saline control group. The results show that theoligonucleotides comprising a GalNAc conjugate group were significantlymore potent than the oligonucleotide lacking a conjugate group.Furthermore, the oligonucleotide comprising a GalNAc conjugate grouplinked to the oligonucleotide via a cleavable moiety (ISIS 680772) waseven more potent than the oligonucleotide comprising a GalNAc conjugategroup linked to the oligonucleotide via a stable moiety (ISIS 696847).

TABLE 112 Modified oligonucleotides targeting mouse APOC-III APOC-IIIISIS Dosage mRNA (% SEQ No. Sequences (5′ to 3′) CM (mg/kg) PBS) ID No.440670 ^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds) n/a 2 92 4906G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)A_(e) 6 86 2059 60 37 680772 GalNAc ₃ -7 _(a-o′) ^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds) PO 0.6 79 4906T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es)A_(es)G_(es)^(m)C_(es)A_(e) 2 58 6 31 20 13 696847 GalNAc ₃ -7 _(a-s′)^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds) n/a (PS)0.6 83 4906 T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es)A_(es)G_(es)^(m)C_(es)A_(e) 2 73 6 40 20 28

The structure of GalNAc₃-7_(a) was shown in Example 48.

Example 102: Distribution in Liver of Antisense OligonucleotidesComprising a GalNAc Conjugate

The liver distribution of ISIS 353382 (see Table 36) that does notcomprise a GalNAc conjugate and ISIS 655861 (see Table 36) that doescomprise a GalNAc conjugate was evaluated. Male balb/c mice weresubcutaneously injected once with ISIS 353382 or 655861 at a dosagelisted in Table 113. Each treatment group consisted of 3 animals exceptfor the 18 mg/kg group for ISIS 655861, which consisted of 2 animals.The animals were sacrificed 48 hours following the dose to determine theliver distribution of the oligonucleotides. In order to measure thenumber of antisense oligonucleotide molecules per cell, a Ruthenium (II)tris-bipyridine tag (MSD TAG, Meso Scale Discovery) was conjugated to anoligonucleotide probe used to detect the antisense oligonucleotides. Theresults presented in Table 113 are the average concentrations ofoligonucleotide for each treatment group in units of millions ofoligonucleotide molecules per cell. The results show that at equivalentdoses, the oligonucleotide comprising a GalNAc conjugate was present athigher concentrations in the total liver and in hepatocytes than theoligonucleotide that does not comprise a GalNAc conjugate. Furthermore,the oligonucleotide comprising a GalNAc conjugate was present at lowerconcentrations in non-parenchymal liver cells than the oligonucleotidethat does not comprise a GalNAc conjugate. And while the concentrationsof ISIS 655861 in hepatocytes and non-parenchymal liver cells weresimilar per cell, the liver is approximately 80% hepatocytes by volume.Thus, the majority of the ISIS 655861 oligonucleotide that was presentin the liver was found in hepatocytes, whereas the majority of the ISIS353382 oligonucleotide that was present in the liver was found innon-parenchymal liver cells.

TABLE 113 Concentration in non- Concentration Concentration parenchymalin whole liver in hepatocytes liver cells Dosage (molecules* (molecules*(molecules* ISIS No. (mg/kg) 10{circumflex over ( )}6 per cell)10{circumflex over ( )}6 per cell) 10{circumflex over ( )}6 per cell)353382 3 9.7 1.2 37.2 10 17.3 4.5 34.0 20 23.6 6.6 65.6 30 29.1 11.780.0 60 73.4 14.8 98.0 90 89.6 18.5 119.9 655861 0.5 2.6 2.9 3.2 1 6.27.0 8.8 3 19.1 25.1 28.5 6 44.1 48.7 55.0 18 76.6 82.3 77.1

Example 103: Duration of Action In Vivo of Oligonucleotides TargetingAPOC-III Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 114 below were tested in a singledose study for duration of action in mice.

TABLE 114 Modified ASOs targeting APOC-III ISIS GalNAc₃ SEQ No.Sequences (5′ to 3′) Cluster CM ID No. 304801 A_(es)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es) n/a n/a 4878T_(es)A_(es)T_(e) 663084 GalNAc ₃ -3 _(a) - _(o′) A _(do)A_(es)G_(eo)^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds)GalNAc₃-3a A_(d) 4894 ^(m)C_(ds)A_(ds)G_(ds)^(m)C_(ds)T_(eo)T_(eo)T_(es)A_(es)T_(e) 679241 A_(es)G_(eo)^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(eo)T_(eo) GalNAc₃-19a A_(d) 4879T_(es)A_(es)T_(eo) A _(do′) -GalNAc ₃ -19 _(a)

The structure of GalNAc₃-3_(a) was shown in Example 39, andGalNAc₃-19_(a) was shown in Example 70.

Treatment

Female transgenic mice that express human APOC-III were each injectedsubcutaneously once with an oligonucleotide listed in Table 114 or withPBS. Each treatment group consisted of 3 animals. Blood was drawn beforedosing to determine baseline and at 3, 7, 14, 21, 28, 35, and 42 daysfollowing the dose. Plasma triglyceride and APOC-III protein levels weremeasured as described in Example 20. The results in Table 115 arepresented as the average percent of plasma triglyceride and APOC-IIIlevels for each treatment group, normalized to baseline levels. Acomparison of the results in Table 71 of example 79 with the results inTable 115 below show that oligonucleotides comprising a mixture ofphosphodiester and phosphorothioate internucleoside linkages exhibitedincreased duration of action than equivalent oligonucleotides comprisingonly phosphorothioate internucleoside linkages.

TABLE 115 Plasma triglyceride and APOC-III protein levels in transgenicmice Time point (days APOC-III ISIS Dosage post- Triglycerides protein(% GalNAc₃ No. (mg/kg) dose) (% baseline) baseline) Cluster CM PBS n/a 396 101 n/a n/a 7 88 98 14 91 103 21 69 92 28 83 81 35 65 86 42 72 88304801 30 3 42 46 n/a n/a 7 42 51 14 59 69 21 67 81 28 79 76 35 72 95 4282 92 663084 10 3 35 28 GalNAc₃-3a A_(d) 7 23 24 14 23 26 21 23 29 28 3022 35 32 36 42 37 47 679241 10 3 38 30 GalNAc₃- A_(d) 7 31 28 19a 14 3022 21 36 34 28 48 34 35 50 45 42 72 64

Example 104: Synthesis of Oligonucleotides Comprising a 5′-GalNAc₂Conjugate

Compound 120 is commercially available, and the synthesis of compound126 is described in Example 49. Compound 120 (1 g, 2.89 mmol), HBTU(0.39 g, 2.89 mmol), and HOBt (1.64 g, 4.33 mmol) were dissolved in DMF(10 mL. and N,N-diisopropylethylamine (1.75 mL, 10.1 mmol) were added.After about 5 min, aminohexanoic acid benzyl ester (1.36 g, 3.46 mmol)was added to the reaction. After 3 h, the reaction mixture was pouredinto 100 mL of 1 μM NaHSO4 and extracted with 2×50 mL ethyl acetate.Organic layers were combined and washed with 3×40 mL sat NaHCO₃ and2×brine, dried with Na₂SO₄, filtered and concentrated. The product waspurified by silica gel column chromatography (DCM:EA:Hex, 1:1:1) toyield compound 231. LCMS and NMR were consistent with the structure.Compounds 231 (1.34 g, 2.438 mmol) was dissolved in dichloromethane (10mL) and trifluoracetic acid (10 mL) was added. After stirring at roomtemperature for 2 h, the reaction mixture was concentrated under reducedpressure and co-evaporated with toluene (3×10 mL). The residue was driedunder reduced pressure to yield compound 232 as the trifuloracetatesalt. The synthesis of compound 166 is described in Example 54. Compound166 (3.39 g, 5.40 mmol) was dissolved in DMF (3 mL). A solution ofcompound 232 (1.3 g, 2.25 mmol) was dissolved in DMF (3 mL) andN,N-diisopropylethylamine (1.55 mL) was added. The reaction was stirredat room temperature for 30 minutes, then poured into water (80 mL) andthe aqueous layer was extracted with EtOAc (2×100 mL). The organic phasewas separated and washed with sat. aqueous NaHCO₃ (3×80 mL), 1 μM NaHSO₄(3×80 mL) and brine (2×80 mL), then dried (Na₂SO₄), filtered, andconcentrated. The residue was purified by silica gel columnchromatography to yield compound 233. LCMS and NMR were consistent withthe structure. Compound 233 (0.59 g, 0.48 mmol) was dissolved inmethanol (2.2 mL) and ethyl acetate (2.2 mL). Palladium on carbon (10 wt% Pd/C, wet, 0.07 g) was added, and the reaction mixture was stirredunder hydrogen atmosphere for 3 h. The reaction mixture was filteredthrough a pad of Celite and concentrated to yield the carboxylic acid.The carboxylic acid (1.32 g, 1.15 mmol, cluster free acid) was dissolvedin DMF (3.2 mL). To this N,N-diisopropylehtylamine (0.3 mL, 1.73 mmol)and PFPTFA (0.30 mL, 1.73 mmol) were added. After 30 min stirring atroom temperature the reaction mixture was poured into water (40 mL) andextracted with EtOAc (2×50 mL). A standard work-up was completed asdescribed above to yield compound 234. LCMS and NMR were consistent withthe structure. Oligonucleotide 235 was prepared using the generalprocedure described in Example 46. The GalNAc₂ cluster portion(GalNAc₂-24_(a)) of the conjugate group GalNAc₂-24 can be combined withany cleavable moiety present on the oligonucleotide to provide a varietyof conjugate groups. The structure of GalNAc₂-24 (GalNAc₂-24_(a)-CM) isshown below:

Example 105: Synthesis of Oligonucleotides Comprising a GalNAc₁-25Conjugate

The synthesis of compound 166 is described in Example 54.Oligonucleotide 236 was prepared using the general procedure describedin Example 46.

Alternatively, oligonucleotide 236 was synthesized using the schemeshown below, and compound 238 was used to form the oligonucleotide 236using procedures described in Example 10.

The GalNAc₁ cluster portion (GalNAc₁-25_(a)) of the conjugate groupGalNAc₁-25 can be combined with any cleavable moiety present on theoligonucleotide to provide a variety of conjugate groups. The structureof GalNAc₁-25 (GalNAc₁-25_(a)-CM) is shown below:

Example 106: Antisense Inhibition In Vivo by Oligonucleotides TargetingSRB-1 Comprising a 5′-GalNAc₂ or a 5′-GalNAc₃ Conjugate

Oligonucleotides listed in Tables 116 and 117 were tested indose-dependent studies for antisense inhibition of SRB-1 in mice.

Treatment

Six to week old, male C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.)were injected subcutaneously once with 2, 7, or 20 mg/kg of ISIS No.440762; or with 0.2, 0.6, 2, 6, or 20 mg/kg of ISIS No. 686221, 686222,or 708561; or with saline. Each treatment group consisted of 4 animals.The mice were sacrificed 72 hours following the final administration.Liver SRB-1 mRNA levels were measured using real-time PCR. SRB-1 mRNAlevels were normalized to cyclophilin mRNA levels according to standardprotocols. The antisense oligonucleotides lowered SRB-1 mRNA levels in adose-dependent manner, and the ED₅₀ results are presented in Tables 116and 117. Although previous studies showed that trivalentGalNAc-conjugated oligonucleotides were significantly more potent thandivalent GalNAc-conjugated oligonucleotides, which were in turnsignificantly more potent than monovalent GalNAc conjugatedoligonucleotides (see, e.g., Khorev et al., Bioorg. & Med. Chem., Vol.16, 5216-5231 (2008)), treatment with antisense oligonucleotidescomprising monovalent, divalent, and trivalent GalNAc clusters loweredSRB-1 mRNA levels with similar potencies as shown in Tables 116 and 117.

TABLE 116 Modified oligonucleotides targeting SRB-1 ISIS ED₅₀ SEQ No.Sequences (5′ to 3′) GalNAc Cluster (mg/kg) ID No 440762 T_(ks)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) n/a 4.7 4880 686221 GalNAc ₂ -24 _(a) -_(o′) A _(do)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) GalNAc₂-24_(a) 0.39 4884^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) 686222 GalNAc ₃ -13 _(a) - _(o′) A_(do)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) GalNAc₃-13_(a) 0.41 4884^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k)See Example 93 for table legend. The structure of GalNAc₃-13a was shownin Example 62, and the structure of GalNAc₂-24a was shown in Example104.

TABLE 117 Modified oligonucleotides targeting SRB-1 ISIS ED₅₀ SEQ No.Sequences (5′ to 3′) GalNAc Cluster (mg/kg) ID No 440762 T_(ks)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) n/a 5 4880 708561 GalNAc ₁ -25 _(a) -_(o′)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) GalNAc₁-25_(a) 0.4 4880^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k)See Example 93 for table legend. The structure of GalNAc₁-25a was shownin Example 105.

The concentrations of the oligonucleotides in Tables 116 and 117 inliver were also assessed, using procedures described in Example 75. Theresults shown in Tables 117a and 117b below are the average totalantisense oligonucleotide tissues levels for each treatment group, asmeasured by UV in units of μg oligonucleotide per gram of liver tissue.The results show that the oligonucleotides comprising a GalNAc conjugategroup accumulated in the liver at significantly higher levels than thesame dose of the oligonucleotide lacking a GalNAc conjugate group.Furthermore, the antisense oligonucleotides comprising one, two, orthree GalNAc ligands in their respective conjugate groups allaccumulated in the liver at similar levels. This result is surprising inview of the Khorev et al. literature reference cited above and isconsistent with the activity data shown in Tables 116 and 117 above.

TABLE 117a Liver concentrations of oligonucleotides comprising a GalNAc₂or GalNAc₃ conjugate group [Antisense Dosage oligonucleotide] GalNAcISIS No. (mg/kg) (μg/g) cluster CM 440762 2 2.1 n/a n/a 7 13.1 20 31.1686221 0.2 0.9 GalNAc₂-24_(a) A_(d) 0.6 2.7 2 12.0 6 26.5 686222 0.2 0.5GalNAc₃-13_(a) A_(d) 0.6 1.6 2 11.6 6 19.8

TABLE 117b Liver concentrations of oligonucleotides comprising a GalNAc₁conjugate group [Antisense Dosage oligonucleotide] GalNAc ISIS No.(mg/kg) (μg/g) cluster CM 440762 2 2.3 n/a n/a 7 8.9 20 23.7 708561 0.20.4 GalNAc₁-25_(a) PO 0.6 1.1 2 5.9 6 23.7 20 53.9

Example 107: Synthesis of Oligonucleotides Comprising a GalNAc₁-26 orGalNAc₁-27 Conjugate

Oligonucleotide 239 is synthesized via coupling of compound 47 (seeExample 15) to acid 64 (see Example 32) using HBTU and DIEA in DMF. Theresulting amide containing compound is phosphitylated, then added to the5′-end of an oligonucleotide using procedures described in Example 10.The GalNAc₁ cluster portion (GalNAc₁-26_(a)) of the conjugate groupGalNAc₁-26 can be combined with any cleavable moiety present on theoligonucleotide to provide a variety of conjugate groups. The structureof GalNAc₁-26 (GalNAc₁-26_(a)-CM) is shown below:

In order to add the GalNAc₁ conjugate group to the 3′-end of anoligonucleotide, the amide formed from the reaction of compounds 47 and64 is added to a solid support using procedures described in Example 7.The oligonucleotide synthesis is then completed using proceduresdescribed in Example 9 in order to form oligonucleotide 240.

The GalNAc₁ cluster portion (GalNAc₁-27_(a)) of the conjugate groupGalNAc₁-27 can be combined with any cleavable moiety present on theoligonucleotide to provide a variety of conjugate groups. The structureof GalNAc₁-27 (GalNAc₁-27_(a)-CM) is shown below:

Example 108: Antisense Inhibition In Vivo by Oligonucleotides Comprisinga GalNAc Conjugate Group Targeting Apo(a) In Vivo

The oligonucleotides listed in Table 118 below were tested in a singledose study in mice.

TABLE 118 Modified ASOs targeting APO(a) ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 494372 T_(es)G_(es) ^(m)C_(es)T_(es)^(m)C_(es) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds)^(m)C_(ds) n/a n/a 4903 T_(ds)T_(es)G_(es)T_(es)T_(es) ^(m)C_(e) 681251GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(es) ^(m)C_(es)T_(es) ^(m)C_(es)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-7a PO 4903 T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(es)G_(es)T_(es)T_(es) ^(m)C_(e) 681255 GalNAc ₃ -3_(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-3a PO 4903 T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e) 681256 GalNAc ₃ -10_(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-10a PO 4903T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e) 681257GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-7a PO 4903 T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e) 681258 GalNAc ₃ -13_(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-13a PO 4903T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e) 681260T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(eo)G_(eo) GalNAc₃-19a A_(d) 4911 T_(es)T_(es)^(m)C_(eo) A _(do′) -GalNAc ₃ -19

The structure of GalNAc₃-7_(a) was shown in Example 48.

Treatment

Male transgenic mice that express human Apo(a) were each injectedsubcutaneously once with an oligonucleotide and dosage listed in Table119 or with PBS. Each treatment group consisted of 4 animals. Blood wasdrawn the day before dosing to determine baseline levels of Apo(a)protein in plasma and at 1 week following the first dose. Additionalblood draws will occur weekly for approximately 8 weeks. Plasma Apo(a)protein levels were measured using an ELISA. The results in Table 119are presented as the average percent of plasma Apo(a) protein levels foreach treatment group, normalized to baseline levels (% BL), The resultsshow that the antisense oligonucleotides reduced Apo(a) proteinexpression. Furthermore, the oligonucleotides comprising a GalNAcconjugate group exhibited even more potent reduction in Apo(a)expression than the oligonucleotide that does not comprise a conjugategroup.

TABLE 119 Apo(a) plasma protein levels Apo(a) at Dosage 1 week ISIS No.(mg/kg) (% BL) PBS n/a 143 494372 50 58 681251 10 15 681255 10 14 68125610 17 681257 10 24 681258 10 22 681260 10 26

Example 109: Synthesis of Oligonucleotides Comprising a GalNAc₁-28 orGalNAc₁-29 Conjugate

Oligonucleotide 241 is synthesized using procedures similar to thosedescribed in Example 71 to form the phosphoramidite intermediate,followed by procedures described in Example 10 to synthesize theoligonucleotide. The GalNAc₁ cluster portion (GalNAc₁-28_(a)) of theconjugate group GalNAc₁-28 can be combined with any cleavable moietypresent on the oligonucleotide to provide a variety of conjugate groups.The structure of GalNAc₁-28 (GalNAc₁-28_(a)-CM) is shown below:

In order to add the GalNAc₁ conjugate group to the 3′-end of anoligonucleotide, procedures similar to those described in Example 71 areused to form the hydroxyl intermediate, which is then added to the solidsupport using procedures described in Example 7. The oligonucleotidesynthesis is then completed using procedures described in Example 9 inorder to form oligonucleotide 242.

The GalNAc₁ cluster portion (GalNAc₁-29_(a)) of the conjugate groupGalNAc₁-29 can be combined with any cleavable moiety present on theoligonucleotide to provide a variety of conjugate groups. The structureof GalNAc₁-29 (GalNAc₁-29_(a)-CM) is shown below:

Example 110: Synthesis of Oligonucleotides Comprising a GalNAc₁-30Conjugate

Oligonucleotide 246 comprising a GalNAc₁-30 conjugate group, wherein Yis selected from O, S, a substituted or unsubstituted C₁-C₁₀ alkyl,amino, substituted amino, azido, alkenyl or alkynyl, is synthesized asshown above. The GalNAc₁ cluster portion (GalNAc₁-30_(a)) of theconjugate group GalNAc₁-30 can be combined with any cleavable moiety toprovide a variety of conjugate groups. In certain embodiments, Y is partof the cleavable moiety. In certain embodiments, Y is part of a stablemoiety, and the cleavable moiety is present on the oligonucleotide. Thestructure of GalNAc₁-30_(a) is shown below:

Example 111: Synthesis of Oligonucleotides Comprising a GalNAc₂-31 orGalNAc₂-32 Conjugate

Oligonucleotide 250 comprising a GalNAc₂-31 conjugate group, wherein Yis selected from O, S, a substituted or unsubstituted C₁-C₁₀ alkyl,amino, substituted amino, azido, alkenyl or alkynyl, is synthesized asshown above. The GalNAc₂ cluster portion (GalNAc₂-31a) of the conjugategroup GalNAc₂-31 can be combined with any cleavable moiety to provide avariety of conjugate groups. In certain embodiments, the Y-containinggroup directly adjacent to the 5′-end of the oligonucleotide is part ofthe cleavable moiety. In certain embodiments, the Y-containing groupdirectly adjacent to the 5′-end of the oligonucleotide is part of astable moiety, and the cleavable moiety is present on theoligonucleotide. The structure of GalNAc₂-31a is shown below:

The synthesis of an oligonucleotide comprising a GalNAc₂-32 conjugate isshown below.

Oligonucleotide 252 comprising a GalNAc₂-32 conjugate group, wherein Yis selected from O, S, a substituted or unsubstituted C₁-C₁₀ alkyl,amino, substituted amino, azido, alkenyl or alkynyl, is synthesized asshown above. The GalNAc₂ cluster portion (GalNAc₂-32_(a)) of theconjugate group GalNAc₂-32 can be combined with any cleavable moiety toprovide a variety of conjugate groups. In certain embodiments, theY-containing group directly adjacent to the 5′-end of theoligonucleotide is part of the cleavable moiety. In certain embodiments,the Y-containing group directly adjacent to the 5′-end of theoligonucleotide is part of a stable moiety, and the cleavable moiety ispresent on the oligonucleotide. The structure of GalNAc₂-32_(a) is shownbelow:

Example 112: Modified Oligonucleotides Comprising a GalNAc₁ Conjugate

The oligonucleotides in Table 120 targeting SRB-1 were synthesized witha GalNAc₁ conjugate group in order to further test the potency ofoligonucleotides comprising conjugate groups that contain one GalNAcligand.

TABLE 120 GalNAc SEQ ISIS No. Sequence (5′ to 3′) cluster CM ID NO.711461 GalNAc ₁ -25 _(a-o′) A _(do) G_(es) ^(m)C_(es) T_(es) T_(es)^(m)C_(es) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) GalNAc₁-25_(a) A_(d)4888 T_(ds) G_(ds) A_(ds) ^(m)C_(ds) T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es)T_(es) T_(e) 711462 GalNAc ₁ -25 _(a-o′)G_(es) ^(m)C_(es) T_(es) T_(es)^(m)C_(es) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) GalNAc₁-25_(a)PO 4886 G_(ds) A_(ds) ^(m)C_(ds) T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es)T_(es) T_(e) 711463 GalNAc ₁ -25 _(a-o′)G_(es) ^(m)C_(eo) T_(eo) T_(eo)^(m)C_(eo) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) GalNAc₁-25_(a)PO 4886 G_(ds) A_(ds) ^(m)C_(ds) T_(ds) T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es) T_(e) 711465 GalNAc ₁ -26 _(a-o′) A _(do) G_(es) ^(m)C_(es)T_(es) T_(es) ^(m)C_(es) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds)GalNAc₁-26_(a) A_(d) 4888 G_(ds) A_(ds) ^(m)C_(ds) T_(ds) T_(es)^(m)C_(es) ^(m)C_(es) T_(es) T_(e) 711466 GalNAc ₁ -26 _(a-o′)G_(es)^(m)C_(es) T_(es) T_(es) ^(m)C_(es) A_(ds) G_(ds) T_(ds) ^(m)C_(ds)A_(ds) T_(ds) GalNAc₁-26_(a) PO 4886 G_(ds) A_(ds) ^(m)C_(ds) T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es) T_(es) T_(e) 711467 GalNAc ₁ -26_(a-o′)G_(es) ^(m)C_(eo) T_(eo) T_(eo) ^(m)C_(eo) A_(ds) G_(ds) T_(ds)^(m)C_(ds) A_(ds) T_(ds) GalNAc₁-26_(a) PO 4886 G_(ds) A_(ds) ^(m)C_(ds)T_(ds) T_(eo) ^(m)C_(eo) ^(m)C_(es) T_(es) T_(e) 711468 GalNAc ₁ -28_(a-o′) A _(do) G_(es) ^(m)C_(es) T_(es) T_(es) ^(m)C_(es) A_(ds) G_(ds)T_(ds) ^(m)C_(ds) A_(ds) GalNAc₁-28_(a) A_(d) 4888 T_(ds) G_(ds) A_(ds)^(m)C_(ds) T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es) T_(es) T_(e) 711469GalNAc ₁ -28 _(a-o′)G_(es) ^(m)C_(es) T_(es) T_(es) ^(m)C_(es) A_(ds)G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) GalNAc₁-28_(a) PO 4886 G_(ds)A_(ds) ^(m)C_(ds) T_(ds) T_(es) ^(m)C_(es) ^(m)C_(es) T_(es) T_(e)711470 GalNAc ₁ -28 _(a-o′)G_(es) ^(m)C_(eo) T_(eo) T_(eo) ^(m)C_(eo)A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) GalNAc₁-28_(a) PO 4886G_(ds) A_(ds) ^(m)C_(ds) T_(ds) T_(eo) ^(m)C_(eo) ^(m)C_(es) T_(es)T_(e) 713844 G_(es) ^(m)C_(es) T_(es) T_(es) ^(m)C_(es) A_(ds) G_(ds)T_(ds) ^(m)C_(ds) A_(ds) T_(ds) G_(ds) A_(ds) ^(m)C_(ds) T_(ds)GalNAc₁-27_(a) PO 4886 T_(es) ^(m)C_(es) ^(m)C_(es) T_(es) T_(eo′-)GalNAc ₁ -27 _(a) 713845 G_(es) ^(m)C_(eo) T_(eo) T_(eo) ^(m)C_(eo)A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) G_(ds) A_(ds) ^(m)C_(ds)T_(ds) GalNAc₁-27_(a) PO 4886 T_(eo) ^(m)C_(eo) ^(m)C_(es) T_(es)T_(eo′-) GalNAc ₁ -27 _(a) 713846 G_(es) ^(m)C_(eo) T_(eo) T_(eo)^(m)C_(eo) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds) G_(ds) A_(ds)^(m)C_(ds) T_(ds) GalNAc₁-27_(a) A_(d) 4887 T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es) T_(eo) A _(do′-) GalNAc ₁ -27 _(a) 713847 G_(es) ^(m)C_(es)T_(es) T_(es) ^(m)C_(es) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds)G_(ds) A_(ds) ^(m)C_(ds) T_(ds) GalNAc₁-29_(a) PO 4886 T_(es) ^(m)C_(es)^(m)C_(es) T_(es) T_(eo′-) GalNAc ₁ -29 _(a) 713848 G_(es) ^(m)C_(eo)T_(eo) T_(eo) ^(m)C_(eo) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds)G_(ds) A_(ds) ^(m)C_(ds) T_(ds) GalNAc₁-29_(a) PO 4886 T_(eo) ^(m)C_(eo)^(m)C_(es) T_(es) T_(eo′-) GalNAc ₁ -29 _(a) 713849 G_(es) ^(m)C_(es)T_(es) T_(es) ^(m)C_(es) A_(ds) G_(ds) T_(ds) ^(m)C_(ds) A_(ds) T_(ds)G_(ds) A_(ds) ^(m)C_(ds) T_(ds) GalNAc₁-29_(a) A_(d) 4887 T_(es)^(m)C_(es) ^(m)C_(es) T_(es) T_(eo) A _(do′-) GalNAc ₁ -29 _(a) 713850G_(es) ^(m)C_(eo) T_(eo) T_(eo) ^(m)C_(eo) A_(ds) G_(ds) T_(ds)^(m)C_(ds) A_(ds) T_(ds) G_(ds) A_(ds) ^(m)C_(ds) T_(ds) GalNAc₁-29_(a)A_(d) 4887 T_(eo) ^(m)C_(eo) ^(m)C_(es) T_(es) T_(eo) A _(do′-) GalNAc ₁-29 _(a)

Example 113: Antisense Oligonucleotides Targeting Angiopoietin-Like 3and Comprising a GalNAc Conjugate Group

The oligonucleotides in Table 121 were designed to target humanangiopoietin-like 3 (ANGPTL3).

TABLE 121 ISIS SEQ No. Sequences (5′ to 3′) ID No. 563580G_(es)G_(es)A_(es) ^(m)C_(es)A_(es)T_(ds)T_(ds)G_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds)G_(ds)T_(ds)A_(ds)A_(ds)T_(es) ^(m)C_(es)G_(es)^(m)C_(es)A_(e) 77 (parent) 658501 G_(es)G_(es)A_(es)^(m)C_(es)A_(es)T_(ds)T_(ds)G_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds)G_(ds)T_(ds)A_(ds)A_(ds)T_(es) ^(m)C_(es)G_(es)^(m)C_(es)A_(eo) A _(do′) -GalNAc ₃ -1 _(a) 4912 666944 GalNAc ₃ -3_(a-o′) A _(do)G_(es)G_(es)A_(es) ^(m)C_(es)A_(es)T_(ds)T_(ds)G_(ds)^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds)T_(ds)A_(ds)A_(ds)T_(es)^(m)C_(es)G_(es) ^(m)C_(es)A_(e) 4913 666945 G_(es)G_(eo)A_(eo)^(m)C_(eo)A_(eo)T_(ds)T_(ds)G_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds)G_(ds)T_(ds)A_(ds)A_(ds)T_(eo) ^(m)C_(eo)G_(es)^(m)C_(es)A_(eo) A _(do′) -GalNAc ₃ -1 _(a) 4912 666946 GalNAc ₃ -3_(a-o′) A _(do)G_(es)G_(eo)A_(eo) ^(m)C_(eo)A_(eo)T_(ds)T_(ds)G_(ds)^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds)T_(ds)A_(ds)A_(ds)T_(eo)^(m)C_(eo)G_(es) ^(m)C_(es)A_(e) 4913 703801 GalNAc ₃ -7_(a-o′)G_(es)G_(es)A_(es) ^(m)C_(es)A_(es)T_(ds)T_(ds)G_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds)G_(ds)T_(ds)A_(ds)A_(ds)T_(es) ^(m)C_(es)G_(es)^(m)C_(es)A_(e) 77 703802 GalNAc ₃ -7 _(a-o′)G_(es)G_(eo)A_(eo)^(m)C_(eo)A_(eo)T_(ds)T_(ds)G_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds)G_(ds)T_(ds)A_(ds)A_(ds)T_(eo) ^(m)C_(eo)G_(es)^(m)C_(es)A_(e) 77

Example 114: Antisense Inhibition In Vivo by Oligonucleotides Comprisinga GalNAc Conjugate Group Targeting Human ANGPTL3

Six week old male, transgenic C57Bl/6 mice that express human ANGPTL3were each injected intraperitoneally once per week at a dosage shownbelow, for a total of two doses, with an oligonucleotide listed in Table122 (and described in Table 121) or with PBS. Each treatment groupconsisted of 4 animals. The mice were sacrificed two days following thefinal dose. ANGPTL3 liver mRNA levels were measured using real-time PCRand RIBOGREEN RNA quantification reagent (Molecular Probes, Inc. Eugene,Oreg.) according to standard protocols. The results below are presentedas the average percent of ANGPTL3 mRNA levels in liver for eachtreatment group, normalized to the PBS control.

As illustrated in Table 122, treatment with antisense oligonucleotideslowered ANGPTL3 liver mRNA levels in a dose-dependent manner, and theoligonucleotide comprising a GalNAc conjugate was significantly morepotent than the parent oligonucleotide lacking a GalNAc conjugate.

TABLE 122 ANGPTL3 liver mRNA levels Dosage mRNA GalNAc₃ ISIS No. (mg/kg)(% PBS) Cluster CM 563580 5 58 n/a n/a 10 56 15 36 25 23 50 20 6585010.3 78 GalNAc₃-1a A_(d) 1 60 3 27 10 19

Liver alanine aminotransferase (ALT) levels were also measured at timeof sacrifice using standard protocols. The results are showed that noneof the treatment groups had elevated ALT levels, indicating that theoligonucleotides were well tolerated.

Example 115: Antisense Inhibition In Vivo by Oligonucleotides Comprisinga GalNAc Conjugate Group Targeting Mouse ANGPTL3

The oligonucleotides listed in Table 123 below were tested in adose-dependent study in mice.

TABLE 123 Modified ASOs targeting mouse ANGPTL3 ISIS GalNAc₃ SEQ No.Sequences (5′ to 3′) Cluster CM ID No. 233693 G_(es)A_(es)^(m)C_(es)A_(es)T_(es)G_(ds)T_(ds)T_(ds) ^(m)C_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(ds) ^(m)C_(ds) n/a n/a 4914 ^(m)C_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es) ^(m)C_(e) 703803 GalNAc ₃ -7 _(a)-_(o′)G_(es)A_(es)^(m)C_(es)A_(es)T_(es)G_(ds)T_(ds)T_(ds) ^(m)C_(ds)T_(ds)T_(ds)GalNAc₃-7a PO 4914 ^(m)C_(ds)A_(ds) ^(m)C_(ds) ^(m)C_(ds)T_(es)^(m)C_(es) ^(m)C_(es)T_(es) ^(m)C_(e) 703804 GalNAc ₃ -7_(a)-_(o′)G_(es)A_(eo) ^(m)C_(eo)A_(eo)T_(eo)G_(ds)T_(ds)T_(ds) ^(m)^(C) _(ds)T_(ds)T_(ds) GalNAc₃-7a PO 4914 ^(m)C_(ds)A_(ds) ^(m)C_(ds)^(m)C_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es) ^(m)C_(e)

The structure of GalNAc₃-7a was shown in Example 48. Low densitylipoprotein receptor knock-out (LDLR^(−/−)) mice were fed a western dietfor 1 week before being injected intraperitoneally once per week at adosage shown below with an oligonucleotide listed in Table 123 or withPBS. Each treatment group consisted of 5 animals. Blood was drawn beforethe first dose was administered in order to determine baseline levelsoftriglycerides in plasma and at 2 weeks following the first dose. Theresults in Table 124 are presented as the average percent of plasmatriglyceride levels for each treatment group, normalized to baselinelevels (% BL), The results show that the antisense oligonucleotidesreduced triglycerides in a dose dependent manner. Furthermore, theoligonucleotides comprising a GalNAc conjugate group exhibited even morepotent reduction in triglycerides than the oligonucleotide that does notcomprise a conjugate group.

TABLE 124 Plasma triglyceride (TG) levels Dosage TG ED₅₀ GalNAc₃ ISISNo. (mg/kg) (% BL) (mg/kg) Cluster CM PBS n/a 110 n/a n/a n/a 233693 192 16 n/a n/a 3 71 10 57 30 42 703803 0.3 96 2 GalNAc₃ ₋ 7a PO 1 69 3 3910 27 703804 0.3 97 2 GalNAc₃ ₋ 7a PO 1 54 3 38 10 26

Example 116: Antisense Inhibition of Human Angiopoietin-Like 3 in Hep3BCells by MOE Gapmers

Antisense oligonucleotides were designed targeting an Angiopoietin-like3 (ANGPTL3) nucleic acid and were tested for their effects on ANGPTL3mRNA in vitro. The antisense oligonucleotides were tested in a series ofexperiments that had similar culture conditions. The results for eachexperiment are presented in separate tables shown below. Cultured Hep3Bcells at a density of 20,000 cells per well were transfected usingelectroporation with 4,500 nM antisense oligonucleotide. After atreatment period of approximately 24 hours, RNA was isolated from thecells and ANGPTL3 mRNA levels were measured by quantitative real-timePCR. Human primer probe set RTS3492_MGB (forward sequenceCCGTGGAAGACCAATATAAACAATT, designated herein as SEQ ID NO: 4;AGTCCTTCTGAGCTGATTTTCTATTTCT; reverse sequence, designated herein as SEQID NO: 5; probe sequence AACCAACAGCATAGTCAAATA, designated herein as SEQID NO: 6) was used to measure mRNA levels. ANGPTL3 mRNA levels wereadjusted according to total RNA content, as measured by RIBOGREEN®.Results are presented as percent inhibition of ANGPTL3, relative tountreated control cells.

The newly designed chimeric antisense oligonucleotides in the Tablesbelow were designed as 5-10-5 MOE gapmers. The 5-10-5 MOE gapmers are 20nucleosides in length, wherein the central gap segment comprises often2′-deoxynucleosides and is flanked by wing segments on the 5′ directionand the 3′ direction comprising five nucleosides each. Each nucleosidein the 5′ wing segment and each nucleoside in the 3′ wing segment has a2′-MOE modification. The internucleoside linkages throughout each gapmerare phosphorothioate (P═S) linkages. All cytosine residues throughouteach gapmer are 5-methylcytosines. “Start site” indicates the 5′-mostnucleoside to which the gapmer is targeted in the human gene sequence.“Stop site” indicates the 3′-most nucleoside to which the gapmer istargeted human gene sequence. Each gapmer listed in the Tables below istargeted to either the human ANGPTL3 mRNA, designated herein as SEQ IDNO: 1 (GENBANK Accession No. NM_014495.2) or the human ANGPTL3 genomicsequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No.NT_032977.9 truncated from nucleotides 33032001 to 33046000). ‘n/a’indicates that the antisense oligonucleotide does not target thatparticular gene sequence with 100% complementarity.

TABLE 125 Inhibition of ANGPTL3 mRNA by 5-10-5 MOE gapmers targeting SEQID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 SEQISIS Start Stop % Start Stop ID NO Site Site Sequence inhibition SiteSite NO 544059 23 42 GATTTTCAATTTCAAGCAAC 40 3127 3146 238 337459 49 68AGCTTAATTGTGAACATTTT 47 3153 3172 239 544060 54 73 GAAGGAGCTTAATTGTGAAC1 3158 3177 240 544061 63 82 CAATAAAAAGAAGGAGCTTA 37 3167 3186 241544062 66 85 GAACAATAAAAAGAAGGAGC 38 3170 3189 242 544063 85 104CTGGAGGAAATAACTAGAGG 30 3189 3208 243 337460 88 107 ATTCTGGAGGAAATAACTAG39 3192 3211 244 544064 112 131 TCAAATGATGAATTGTCTTG 36 3216 3235 245544065 138 157 TTGATTTTGGCTCTGGAGAT 26 3242 3261 246 544066 145 164GCAAATCTTGATTTTGGCTC 56 3249 3268 247 233676 148 167ATAGCAAATCTTGATTTTGG 69 3252 3271 248 544067 156 175CGTCTAACATAGCAAATCTT 64 3260 3279 249 544068 174 193TGGCTAAAATTTTTACATCG 28 3278 3297 250 544069 178 197CCATTGGCTAAAATTTTTAC 0 3282 3301 251 544070 184 203 AGGAGGCCATTGGCTAAAAT7 3288 3307 252 544071 187 206 TGAAGGAGGCCATTGGCTAA 32 3291 3310 253544072 195 214 GTCCCAACTGAAGGAGGCCA 9 3299 3318 254 544073 199 218CCATGTCCCAACTGAAGGAG 6 3303 3322 255 544074 202 221 AGACCATGTCCCAACTGAAG18 3306 3325 256 544075 206 225 TTTAAGACCATGTCCCAACT 0 3310 3329 257544076 209 228 GTCTTTAAGACCATGTCCCA 0 3313 3332 258 544077 216 235GGACAAAGTCTTTAAGACCA 0 3320 3339 259 544078 222 241 TCTTATGGACAAAGTCTTTA0 3326 3345 260 544079 245 264 TATGTCATTAATTTGGCCCT 0 3349 3368 261544080 270 289 GATCAAATATGTTGAGTTTT 27 3374 3393 262 233690 274 293GACTGATCAAATATGTTGAG 49 3378 3397 263 544081 316 335TCTTCTTTGATTTCACTGGT 62 3420 3439 264 544082 334 353CTTCTCAGTTCCTTTTCTTC 35 3438 3457 265 544083 337 356GTTCTTCTCAGTTCCTTTTC 60 3441 3460 266 544084 341 360TGTAGTTCTTCTCAGTTCCT 51 3445 3464 267 544431 345 364TATATGTAGTTCTTCTCAGT 9 3449 3468 268 544086 348 367 GTTTATATGTAGTTCTTCTC39 3452 3471 269 544087 352 371 TGTAGTTTATATGTAGTTCT 30 3456 3475 270544088 356 375 GACTTGTAGTTTATATGTAG 12 3460 3479 271 544089 364 383TCATTTTTGACTTGTAGTTT 31 3468 3487 272 544090 369 388CCTCTTCATTTTTGACTTGT 61 3473 3492 273 544091 375 394TCTTTACCTCTTCATTTTTG 48 3479 3498 274 544092 380 399CATATTCTTTACCTCTTCAT 35 3484 3503 275 544093 384 403GTGACATATTCTTTACCTCT 63 3488 3507 276 544094 392 411GAGTTCAAGTGACATATTCT 53 3496 3515 277 544095 398 417TGAGTTGAGTTCAAGTGACA 31 3502 3521 278 544096 403 422AGTTTTGAGTTGAGTTCAAG 14 3507 3526 279 544097 406 425TCAAGTTTTGAGTTGAGTTC 38 3510 3529 280 544098 414 433GGAGGCTTTCAAGTTTTGAG 39 3518 3537 281 544099 423 442TTTCTTCTAGGAGGCTTTCA 57 3527 3546 282 544100 427 446ATTTTTTCTTCTAGGAGGCT 39 3531 3550 283 544101 432 451GTAGAATTTTTTCTTCTAGG 28 3536 3555 284 544102 462 481GCTCTTCTAAATATTTCACT 60 3566 3585 285 544103 474 493AGTTAGTTAGTTGCTCTTCT 40 3578 3597 286 544104 492 511CAGGTTGATTTTGAATTAAG 38 3596 3615 287 544105 495 514TTTCAGGTTGATTTTGAATT 28 3599 3618 288 544106 499 518GGAGTTTCAGGTTGATTTTG 38 3603 3622 289 544107 504 523GTTCTGGAGTTTCAGGTTGA 50 3608 3627 290 544108 526 545TTAAGTGAAGTTACTTCTGG 20 3630 3649 291 544109 555 574TGCTATTATCTTGTTTTTCT 23 4293 4312 292 544110 564 583GGTCTTTGATGCTATTATCT 67 4302 4321 293 544111 567 586GAAGGTCTTTGATGCTATTA 49 4305 4324 294 544112 572 591CTGGAGAAGGTCTTTGATGC 52 4310 4329 295 544113 643 662CTGAGCTGATTTTCTATTTC 12 n/a n/a 296 337477 664 683 GGTTCTTGAATACTAGTCCT70 6677 6696 234 544114 673 692 ATTTCTGTGGGTTCTTGAAT 32 6686 6705 297337478 675 694 AAATTTCTGTGGGTTCTTGA 51 6688 6707 235 544115 678 697GAGAAATTTCTGTGGGTTCT 54 6691 6710 298 544116 682 701GATAGAGAAATTTCTGTGGG 25 6695 6714 299 544117 689 708CTTGGAAGATAGAGAAATTT 16 6702 6721 300 337479 692 711TGGCTTGGAAGATAGAGAAA 34 6705 6724 236 544118 699 718GTGCTCTTGGCTTGGAAGAT 64 6712 6731 301 544119 703 722CTTGGTGCTCTTGGCTTGGA 70 6716 6735 302 544120 707 726AGTTCTTGGTGCTCTTGGCT 82 6720 6739 15 233710 710 729 AGTAGTTCTTGGTGCTCTTG63 6723 6742 233 544121 713 732 GGGAGTAGTTCTTGGTGCTC 64 6726 6745 303544122 722 741 CTGAAGAAAGGGAGTAGTTC 24 6735 6754 304 544123 752 771ATCATGTTTTACATTTCTTA 0 6765 6784 305 544124 755 774 GCCATCATGTTTTACATTTC35 n/a n/a 306 544125 759 778 GAATGCCATCATGTTTTACA 8 n/a n/a 307 544126762 781 CAGGAATGCCATCATGTTTT 6 n/a n/a 308 337487 804 823CACTTGTATGTTCACCTCTG 65 7389 7408 28 233717 889 908 TGAATTAATGTCCATGGACT33 7876 7895 14

TABLE 126 Inhibition of ANGPTL3 mRNA by 5-10-5 MOE gapmers targeting SEQID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: 1 NO: 2 NO: 2 SEQISIS Start Stop % Start Stop ID NO Site Site Sequence inhibition SiteSite NO 544204 n/a n/a GACTTCTTAACTCTATATAT 0 3076 3095 309 544205 n/an/a CTAGACTTCTTAACTCTATA 0 3079 3098 310 544206 n/a n/aGACCTAGACTTCTTAACTCT 0 3082 3101 311 544207 n/a n/a GGAAGCAGACCTAGACTTCT21 3089 3108 312 544208 n/a n/a TCTGGAAGCAGACCTAGACT 23 3092 3111 313544209 n/a n/a TCTTCTGGAAGCAGACCTAG 7 3095 3114 314 544210 n/a n/aCTAATCTTTAGGGATTTAGG 24 11433 11452 315 544211 n/a n/aTGTATCTAATCTTTAGGGAT 2 11438 11457 316 544213 n/a n/aTAACTTGGGCACTATATCCT 44 11553 11572 317 544214 n/a n/aATTGACAAAGGTAGGTCACC 59 11576 11595 318 544215 n/a n/aATATGACATGTATATTGGAT 41 11620 11639 319 544216 n/a n/aTTTTGTACTTTTCTGGAACA 34 11704 11723 320 544217 n/a n/aTAGTCTGTGGTCCTGAAAAT 32 11748 11767 321 544218 n/a n/aAGCTTAGTCTGTGGTCCTGA 20 11752 11771 322 544219 n/a n/aGACAGCTTAGTCTGTGGTCC 45 11755 11774 323 544220 n/a n/aGTATTCTGGCCCTAAAAAAA 2 11789 11808 324 544221 n/a n/aATTTTGGTATTCTGGCCCTA 39 11795 11814 325 544223 n/a n/aTTTGCATTTGAAATTGTCCA 32 11837 11856 326 544224 n/a n/aGGAAGCAACTCATATATTAA 39 11869 11888 327 544225 n/a n/aTATCAGAAAAAGATACCTGA 0 9821 9840 328 544226 n/a n/a ATAATAGCTAATAATGTGGG15 9875 9894 329 544227 n/a n/a TGCAGATAATAGCTAATAAT 31 9880 9899 330544228 n/a n/a TGTCATTGCAGATAATAGCT 61 9886 9905 331 544229 n/a n/aTAAAAGTTGTCATTGCAGAT 38 9893 9912 332 544230 n/a n/aCGGATTTTTAAAAGTTGTCA 45 9901 9920 333 544231 n/a n/aGGGATTCGGATTTTTAAAAG 0 9907 9926 334 544232 n/a n/a TTTGGGATTCGGATTTTTAA24 9910 9929 335 544233 n/a n/a ACGCTTATTTGGGATTCGGA 53 9917 9936 336544251 n/a n/a TTTAAGAGATTTACAAGTCA 11 2811 2830 337 544252 n/a n/aGACTACCTGTTTTTAAAAGC 6 2851 2870 338 544253 n/a n/a TATGGTGACTACCTGTTTTT12 2857 2876 339 544254 n/a n/a ACTTTGCTGTATTATAAACT 12 2890 2909 340544255 n/a n/a ATTGTATTTAACTTTGCTGT 0 2900 2919 341 544256 n/a n/aGAGCAACTAACTTAATAGGT 13 2928 2947 342 544257 n/a n/aGAAATGAGCAACTAACTTAA 25 2933 2952 343 544258 n/a n/aAATCAAAGAAATGAGCAACT 0 2940 2959 344 544259 n/a n/a ACCTTCTTCCACATTGAGTT8 2977 2996 345 544260 n/a n/a CACGAATGTAACCTTCTTCC 0 2987 3006 346544261 n/a n/a TTAACTTGCACGAATGTAAC 27 2995 3014 347 544262 n/a n/aTATATATACCAATATTTGCC 0 3063 3082 348 544263 n/a n/a TCTTAACTCTATATATACCA0 3072 3091 349 544264 n/a n/a CTTTAAGTGAAGTTACTTCT 17 3632 3651 350544265 n/a n/a TCTACTTACTTTAAGTGAAG 9 3640 3659 351 544266 n/a n/aGAACCCTCTTTATTTTCTAC 1 3655 3674 352 544267 n/a n/a ACATAAACATGAACCCTCTT6 3665 3684 353 544268 n/a n/a CCACATTGAAAACATAAACA 25 3676 3695 354544269 n/a n/a GCATGCCTTAGAAATATTTT 7 3707 3726 355 544270 n/a n/aCAATGCAACAAAGTATTTCA 0 3731 3750 356 544271 n/a n/a CTGGAGATTATTTTTCTTGG34 3768 3787 357 544272 n/a n/a TTCATATATAACATTAGGGA 0 3830 3849 358544273 n/a n/a TCAGTGTTTTCATATATAAC 18 3838 3857 359 544274 n/a n/aGACATAGTGTTCTAGATTGT 14 3900 3919 360 544275 n/a n/aCAATAGTGTAATGACATAGT 21 3912 3931 361 544276 n/a n/aTTACTTACCTTCAGTAATTT 0 3933 3952 362 544277 n/a n/a ATCTTTTCCATTTACTGTAT8 4005 4024 363 544278 n/a n/a AGAAAAAGCCCAGCATATTT 11 4037 4056 364544279 n/a n/a GTATGCTTCTTTCAAATAGC 36 4130 4149 365 544280 n/a n/aCCTTCCCCTTGTATGCTTCT 41 4140 4159 366 544281 n/a n/aCCTGTAACACTATCATAATC 1 4207 4226 367 544282 n/a n/a TGACTTACCTGATTTTCTAT6 4384 4403 368 544283 n/a n/a GATGGGACATACCATTAAAA 0 4407 4426 369544284 n/a n/a GTGAAAGATGGGACATACCA 20 4413 4432 370 544285 n/a n/aCCTGTGTGAAAGATGGGACA 6 4418 4437 371 544286 n/a n/a CATTGGCTGCTATGAATTAA41 4681 4700 372 544287 n/a n/a GATGACATTGGCTGCTATGA 40 4686 4705 373544288 n/a n/a GAGAAACATGATCTAATTTG 12 4717 4736 374 544289 n/a n/aATGGAAAGCTATTGTGTGGT 0 4747 4766 375 544290 n/a n/a GTCTAAAGAGCCAATATGAG22 4771 4790 376 544291 n/a n/a AATCTTGGTCTAAAGAGCCA 46 4778 4797 377544433 n/a n/a GAGATTTACAAGTCAAAAAT 4 2806 2825 378 544434 n/a n/aATTTAACTTTGCTGTATTAT 0 2895 2914 379 544435 n/a n/a ATCAATGCTAAATGAAATCA0 2955 2974 380 544436 n/a n/a TATTTTCTGGAGATTATTTT 0 3774 3793 381544437 n/a n/a AAAATGAATATTGGCAATTC 0 4159 4178 382 233717 889 908TGAATTAATGTCCATGGACT 36 7876 7895 14 544202 2081 2100AAAGTCAATGTGACTTAGTA 42 11053 11072 383 544203 2104 2123AAGGTATAGTGATACCTCAT 56 11076 11095 384

TABLE 127 Inhibition of ANGPTL3 mRNA by 5-10-5 MOE gapmers targeting SEQID NO: 1 and 2 SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: NO: 2 NO: 2 SEQISIS Start 1 Stop % Start Stop ID NO Site Site Sequence inhibition SiteSite NO 544127 765 784 CAGCAGGAATGCCATCATGT 4 N/A N/A 385 544128 819 838TGATGGCATACATGCCACTT 0 7404 7423 386 544129 828 847 TGCTGGGTCTGATGGCATAC44 7413 7432 387 544130 832 851 GAGTTGCTGGGTCTGATGGC 16 7417 7436 388544131 841 860 AAAACTTGAGAGTTGCTGGG 0 7426 7445 389 544132 848 867GACATGAAAAACTTGAGAGT 0 7433 7452 390 544133 859 878 ACATCACAGTAGACATGAAA25 7444 7463 391 233717 889 908 TGAATTAATGTCCATGGACT 36 7876 7895 14544134 915 934 AGTTTTGTGATCCATCTATT 46 7902 7921 392 544135 918 937TGAAGTTTTGTGATCCATCT 42 7905 7924 393 544136 926 945CGTTTCATTGAAGTTTTGTG 45 7913 7932 394 544137 946 965CCATATTTGTAGTTCTCCCA 44 7933 7952 395 544138 949 968AAACCATATTTGTAGTTCTC 25 7936 7955 396 544139 970 989AATTCTCCATCAAGCCTCCC 35 N/A N/A 397 233722 991 1010 ATCTTCTCTAGGCCCAACCA65 9566 9585 398 544432 997 1016 GAGTATATCTTCTCTAGGCC 0 9572 9591 399544140 1002 1021 CTATGGAGTATATCTTCTCT 6 9577 9596 400 544141 1008 1027GCTTCACTATGGAGTATATC 63 9583 9602 401 544142 1013 1032AGATTGCTTCACTATGGAGT 52 9588 9607 402 544143 1046 1065CCAGTCTTCCAACTCAATTC 35 9621 9640 403 544144 1052 1071GTCTTTCCAGTCTTCCAACT 64 9627 9646 404 544145 1055 1074GTTGTCTTTCCAGTCTTCCA 80 9630 9649 16 544146 1059 1078GTTTGTTGTCTTTCCAGTCT 59 9634 9653 405 544147 1062 1081AATGTTTGTTGTCTTTCCAG 12 9637 9656 406 544148 1095 1114CGTGATTTCCCAAGTAAAAA 56 9670 9689 407 544149 1160 1179GTTTTCCGGGATTGCATTGG 33 9735 9754 408 544150 1165 1184TCTTTGTTTTCCGGGATTGC 54 9740 9759 409 544151 1170 1189CCAAATCTTTGTTTTCCGGG 64 9745 9764 410 544152 1173 1192ACACCAAATCTTTGTTTTCC 37 9748 9767 411 544153 1178 1197AGAAAACACCAAATCTTTGT 32 9753 9772 412 544154 1183 1202CAAGTAGAAAACACCAAATC 13 9758 9777 413 544155 1188 1207GATCCCAAGTAGAAAACACC 0 9763 9782 414 544156 1195 1214GCTTTGTGATCCCAAGTAGA 74 9770 9789 17 544157 1198 1217TTTGCTTTGTGATCCCAAGT 73 9773 9792 415 544158 1202 1221TCCTTTTGCTTTGTGATCCC 62 9777 9796 416 544159 1208 1227GAAGTGTCCTTTTGCTTTGT 30 9783 9802 417 544160 1246 1265TGCCACCACCAGCCTCCTGA 60 N/A N/A 418 544161 1253 1272CTCATCATGCCACCACCAGC 73 10225 10244 419 544162 1269 1288GGTTGTTTTCTCCACACTCA 76 10241 10260 18 544163 1276 1295CCATTTAGGTTGTTTTCTCC 25 10248 10267 420 544164 1283 1302ATATTTACCATTTAGGTTGT 25 10255 10274 421 544165 1294 1313CTTGGTTTGTTATATTTACC 63 10266 10285 422 544166 1353 1372ACCTTCCATTTTGAGACTTC 75 10325 10344 19 544167 1363 1382ATAGAGTATAACCTTCCATT 71 10335 10354 423 544168 1367 1386TTTTATAGAGTATAACCTTC 37 10339 10358 424 544169 1374 1393TGGTTGATTTTATAGAGTAT 37 10346 10365 425 544170 1378 1397ATTTTGGTTGATTTTATAGA 3 10350 10369 426 544171 1383 1402TCAACATTTTGGTTGATTTT 16 10355 10374 427 544172 1390 1409GGATGGATCAACATTTTGGT 51 10362 10381 428 544173 1393 1412GTTGGATGGATCAACATTTT 62 10365 10384 429 544174 1396 1415TCTGTTGGATGGATCAACAT 5 10368 10387 430 544175 1401 1420CTGAATCTGTTGGATGGATC 55 10373 10392 431 544176 1407 1426AGCTTTCTGAATCTGTTGGA 65 10379 10398 432 544177 1414 1433CATTCAAAGCTTTCTGAATC 21 10386 10405 433 544178 1417 1436GTTCATTCAAAGCTTTCTGA 66 10389 10408 434 544179 1420 1439TCAGTTCATTCAAAGCTTTC 6 10392 10411 435 544180 1423 1442GCCTCAGTTCATTCAAAGCT 68 10395 10414 436 544181 1427 1446ATTTGCCTCAGTTCATTCAA 53 10399 10418 437 544182 1431 1450TTAAATTTGCCTCAGTTCAT 40 10403 10422 438 544183 1436 1455GCCTTTTAAATTTGCCTCAG 70 10408 10427 439 544184 1498 1517AGGATTTAATACCAGATTAT 38 10470 10489 440 544185 1502 1521CTTAAGGATTTAATACCAGA 56 10474 10493 441 544186 1505 1524TCTCTTAAGGATTTAATACC 33 10477 10496 442 544187 1546 1565GACAGTGACTTTAAGATAAA 35 10518 10537 443 544188 1572 1591TGTGATTGTATGTTTAATCT 48 10544 10563 444 544189 1578 1597AGGTTATGTGATTGTATGTT 48 10550 10569 445 544190 1583 1602CTTTAAGGTTATGTGATTGT 48 10555 10574 446 544191 1589 1608GGTATTCTTTAAGGTTATGT 62 10561 10580 447 544192 1656 1675ATTGATTCCCACATCACAAA 47 10628 10647 448 544193 1661 1680CTAAAATTGATTCCCACATC 67 10633 10652 449 544194 1665 1684CCATCTAAAATTGATTCCCA 63 10637 10656 450 544195 1771 1790TTGTGATATTAGCTCATATG 59 10743 10762 451 544196 1794 1813ACTAGTTTTTTAAACTGGGA 28 10766 10785 452 544197 1820 1839GTCAAGTTTAGAGTTTTAAC 44 10792 10811 453 544198 1826 1845TATTTAGTCAAGTTTAGAGT 14 10798 10817 454 544199 1907 1926TACACATACTCTGTGCTGAC 82 10879 10898 20 544200 1913 1932GATTTTTACACATACTCTGT 57 10885 10904 455 544201 2008 2027CTGCTTCATTAGGTTTCATA 61 10980 10999 456

TABLE 128 Inhibition of ANGPTL3 mRNA by 5-10-5 MOE gapmers targeting SEQID NO: 1 and 2 SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: NO: 2 NO: 2 ISISStart 1 Stop % Start Stop SEQ ID NO Site Site Sequence inhibition SiteSite NO 544127 765 784 CAGCAGGAATGCCATCATGT 0 N/A N/A 457 544128 819 838TGATGGCATACATGCCACTT 13 7404 7423 458 544129 828 847TGCTGGGTCTGATGGCATAC 49 7413 7432 459 544130 832 851GAGTTGCTGGGTCTGATGGC 27 7417 7436 460 544131 841 860AAAACTTGAGAGTTGCTGGG 0 7426 7445 461 544132 848 867 GACATGAAAAACTTGAGAGT0 7433 7452 462 544133 859 878 ACATCACAGTAGACATGAAA 18 7444 7463 463233717 889 908 TGAATTAATGTCCATGGACT 55 7876 7895 14 544134 915 934AGTTTTGTGATCCATCTATT 68 7902 7921 464 544135 918 937TGAAGTTTTGTGATCCATCT 77 7905 7924 465 544136 926 945CGTTTCATTGAAGTTTTGTG 60 7913 7932 466 544137 946 965CCATATTTGTAGTTCTCCCA 64 7933 7952 467 544138 949 968AAACCATATTTGTAGTTCTC 45 7936 7955 468 544139 970 989AATTCTCCATCAAGCCTCCC 70 N/A N/A 469 233722 991 1010 ATCTTCTCTAGGCCCAACCA96 9566 9585 470 544432 997 1016 GAGTATATCTTCTCTAGGCC 69 9572 9591 471544140 1002 1021 CTATGGAGTATATCTTCTCT 37 9577 9596 472 544141 1008 1027GCTTCACTATGGAGTATATC 65 9583 9602 473 544142 1013 1032AGATTGCTTCACTATGGAGT 55 9588 9607 474 544143 1046 1065CCAGTCTTCCAACTCAATTC 31 9621 9640 475 544144 1052 1071GTCTTTCCAGTCTTCCAACT 72 9627 9646 476 544145 1055 1074GTTGTCTTTCCAGTCTTCCA 86 9630 9649 16 544146 1059 1078GTTTGTTGTCTTTCCAGTCT 66 9634 9653 477 544147 1062 1081AATGTTTGTTGTCTTTCCAG 21 9637 9656 478 544148 1095 1114CGTGATTTCCCAAGTAAAAA 63 9670 9689 479 544149 1160 1179GTTTTCCGGGATTGCATTGG 32 9735 9754 480 544150 1165 1184TCTTTGTTTTCCGGGATTGC 48 9740 9759 481 544151 1170 1189CCAAATCTTTGTTTTCCGGG 72 9745 9764 482 544152 1173 1192ACACCAAATCTTTGTTTTCC 39 9748 9767 483 544153 1178 1197AGAAAACACCAAATCTTTGT 39 9753 9772 484 544154 1183 1202CAAGTAGAAAACACCAAATC 22 9758 9777 485 544155 1188 1207GATCCCAAGTAGAAAACACC 5 9763 9782 486 544156 1195 1214GCTTTGTGATCCCAAGTAGA 79 9770 9789 17 544157 1198 1217TTTGCTTTGTGATCCCAAGT 80 9773 9792 487 544158 1202 1221TCCTTTTGCTTTGTGATCCC 73 9777 9796 488 544159 1208 1227GAAGTGTCCTTTTGCTTTGT 33 9783 9802 489 544160 1246 1265TGCCACCACCAGCCTCCTGA 67 N/A N/A 490 544161 1253 1272CTCATCATGCCACCACCAGC 79 10225 10244 491 544162 1269 1288GGTTGTTTTCTCCACACTCA 84 10241 10260 18 544163 1276 1295CCATTTAGGTTGTTTTCTCC 34 10248 10267 492 544164 1283 1302ATATTTACCATTTAGGTTGT 17 10255 10274 493 544165 1294 1313CTTGGTTTGTTATATTTACC 76 10266 10285 494 544166 1353 1372ACCTTCCATTTTGAGACTTC 79 10325 10344 19 544167 1363 1382ATAGAGTATAACCTTCCATT 73 10335 10354 495 544168 1367 1386TTTTATAGAGTATAACCTTC 41 10339 10358 496 544169 1374 1393TGGTTGATTTTATAGAGTAT 53 10346 10365 497 544170 1378 1397ATTTTGGTTGATTTTATAGA 28 10350 10369 498 544171 1383 1402TCAACATTTTGGTTGATTTT 19 10355 10374 499 544172 1390 1409GGATGGATCAACATTTTGGT 66 10362 10381 500 544173 1393 1412GTTGGATGGATCAACATTTT 71 10365 10384 501 544174 1396 1415TCTGTTGGATGGATCAACAT 35 10368 10387 502 544175 1401 1420CTGAATCTGTTGGATGGATC 68 10373 10392 503 544176 1407 1426AGCTTTCTGAATCTGTTGGA 70 10379 10398 504 544177 1414 1433CATTCAAAGCTTTCTGAATC 35 10386 10405 505 544178 1417 1436GTTCATTCAAAGCTTTCTGA 76 10389 10408 506 544179 1420 1439TCAGTTCATTCAAAGCTTTC 15 10392 10411 507 544180 1423 1442GCCTCAGTTCATTCAAAGCT 68 10395 10414 508 544181 1427 1446ATTTGCCTCAGTTCATTCAA 67 10399 10418 509 544182 1431 1450TTAAATTTGCCTCAGTTCAT 51 10403 10422 510 544183 1436 1455GCCTTTTAAATTTGCCTCAG 80 10408 10427 511 544184 1498 1517AGGATTTAATACCAGATTAT 54 10470 10489 512 544185 1502 1521CTTAAGGATTTAATACCAGA 69 10474 10493 513 544186 1505 1524TCTCTTAAGGATTTAATACC 58 10477 10496 514 544187 1546 1565GACAGTGACTTTAAGATAAA 34 10518 10537 515 544188 1572 1591TGTGATTGTATGTTTAATCT 47 10544 10563 516 544189 1578 1597AGGTTATGTGATTGTATGTT 68 10550 10569 517 544190 1583 1602CTTTAAGGTTATGTGATTGT 62 10555 10574 518 544191 1589 1608GGTATTCTTTAAGGTTATGT 66 10561 10580 519 544192 1656 1675ATTGATTCCCACATCACAAA 50 10628 10647 520 544193 1661 1680CTAAAATTGATTCCCACATC 73 10633 10652 521 544194 1665 1684CCATCTAAAATTGATTCCCA 73 10637 10656 522 544195 1771 1790TTGTGATATTAGCTCATATG 57 10743 10762 523 544196 1794 1813ACTAGTTTTTTAAACTGGGA 21 10766 10785 524 544197 1820 1839GTCAAGTTTAGAGTTTTAAC 53 10792 10811 525 544198 1826 1845TATTTAGTCAAGTTTAGAGT 11 10798 10817 526 544199 1907 1926TACACATACTCTGTGCTGAC 84 10879 10898 20 544200 1913 1932GATTTTTACACATACTCTGT 53 10885 10904 527 544201 2008 2027CTGCTTCATTAGGTTTCATA 67 10980 10999 528

TABLE 129 Inhibition of ANGPTL3 mRNA by 5-10-5 MOE gapmers targeting SEQID NO: 1 and 2 SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: NO: 2 NO: 2 ISISStart 1 Stop % Start Stop SEQ ID NO Site Site Sequence inhibition SiteSite NO 544127 765 784 CAGCAGGAATGCCATCATGT 18 N/A N/A 529 544128 819838 TGATGGCATACATGCCACTT 0 7404 7423 530 544129 828 847TGCTGGGTCTGATGGCATAC 48 7413 7432 531 544130 832 851GAGTTGCTGGGTCTGATGGC 14 7417 7436 532 544131 841 860AAAACTTGAGAGTTGCTGGG 5 7426 7445 533 544132 848 867 GACATGAAAAACTTGAGAGT0 7433 7452 534 544133 859 878 ACATCACAGTAGACATGAAA 28 7444 7463 535233717 889 908 TGAATTAATGTCCATGGACT 51 7876 7895 14 544134 915 934AGTTTTGTGATCCATCTATT 36 7902 7921 536 544135 918 937TGAAGTTTTGTGATCCATCT 61 7905 7924 537 544136 926 945CGTTTCATTGAAGTTTTGTG 54 7913 7932 538 544137 946 965CCATATTTGTAGTTCTCCCA 67 7933 7952 539 544138 949 968AAACCATATTTGTAGTTCTC 39 7936 7955 540 544139 970 989AATTCTCCATCAAGCCTCCC 77 N/A N/A 541 233722 991 1010 ATCTTCTCTAGGCCCAACCA95 9566 9585 542 544432 997 1016 GAGTATATCTTCTCTAGGCC 86 9572 9591 543544140 1002 1021 CTATGGAGTATATCTTCTCT 57 9577 9596 544 544141 1008 1027GCTTCACTATGGAGTATATC 52 9583 9602 545 544142 1013 1032AGATTGCTTCACTATGGAGT 40 9588 9607 546 544143 1046 1065CCAGTCTTCCAACTCAATTC 32 9621 9640 547 544144 1052 1071GTCTTTCCAGTCTTCCAACT 53 9627 9646 548 544145 1055 1074GTTGTCTTTCCAGTCTTCCA 80 9630 9649 16 544146 1059 1078GTTTGTTGTCTTTCCAGTCT 59 9634 9653 549 544147 1062 1081AATGTTTGTTGTCTTTCCAG 42 9637 9656 550 544148 1095 1114CGTGATTTCCCAAGTAAAAA 76 9670 9689 551 544149 1160 1179GTTTTCCGGGATTGCATTGG 29 9735 9754 552 544150 1165 1184TCTTTGTTTTCCGGGATTGC 50 9740 9759 553 544151 1170 1189CCAAATCTTTGTTTTCCGGG 56 9745 9764 554 544152 1173 1192ACACCAAATCTTTGTTTTCC 26 9748 9767 555 544153 1178 1197AGAAAACACCAAATCTTTGT 22 9753 9772 556 544154 1183 1202CAAGTAGAAAACACCAAATC 29 9758 9777 557 544155 1188 1207GATCCCAAGTAGAAAACACC 16 9763 9782 558 544156 1195 1214GCTTTGTGATCCCAAGTAGA 71 9770 9789 17 544157 1198 1217TTTGCTTTGTGATCCCAAGT 55 9773 9792 559 544158 1202 1221TCCTTTTGCTTTGTGATCCC 51 9777 9796 560 544159 1208 1227GAAGTGTCCTTTTGCTTTGT 8 9783 9802 561 544160 1246 1265TGCCACCACCAGCCTCCTGA 68 N/A N/A 562 544161 1253 1272CTCATCATGCCACCACCAGC 48 10225 10244 563 544162 1269 1288GGTTGTTTTCTCCACACTCA 74 10241 10260 18 544163 1276 1295CCATTTAGGTTGTTTTCTCC 33 10248 10267 564 544164 1283 1302ATATTTACCATTTAGGTTGT 0 10255 10274 565 544165 1294 1313CTTGGTTTGTTATATTTACC 52 10266 10285 566 544166 1353 1372ACCTTCCATTTTGAGACTTC 69 10325 10344 19 544167 1363 1382ATAGAGTATAACCTTCCATT 72 10335 10354 567 544168 1367 1386TTTTATAGAGTATAACCTTC 27 10339 10358 568 544169 1374 1393TGGTTGATTTTATAGAGTAT 39 10346 10365 569 544170 1378 1397ATTTTGGTTGATTTTATAGA 7 10350 10369 570 544171 1383 1402TCAACATTTTGGTTGATTTT 0 10355 10374 571 544172 1390 1409GGATGGATCAACATTTTGGT 48 10362 10381 572 544173 1393 1412GTTGGATGGATCAACATTTT 51 10365 10384 573 544174 1396 1415TCTGTTGGATGGATCAACAT 46 10368 10387 574 544175 1401 1420CTGAATCTGTTGGATGGATC 58 10373 10392 575 544176 1407 1426AGCTTTCTGAATCTGTTGGA 57 10379 10398 576 544177 1414 1433CATTCAAAGCTTTCTGAATC 0 10386 10405 577 544178 1417 1436GTTCATTCAAAGCTTTCTGA 62 10389 10408 578 544179 1420 1439TCAGTTCATTCAAAGCTTTC 21 10392 10411 579 544180 1423 1442GCCTCAGTTCATTCAAAGCT 73 10395 10414 580 544181 1427 1446ATTTGCCTCAGTTCATTCAA 46 10399 10418 581 544182 1431 1450TTAAATTTGCCTCAGTTCAT 52 10403 10422 582 544183 1436 1455GCCTTTTAAATTTGCCTCAG 66 10408 10427 583 544184 1498 1517AGGATTTAATACCAGATTAT 31 10470 10489 584 544185 1502 1521CTTAAGGATTTAATACCAGA 49 10474 10493 585 544186 1505 1524TCTCTTAAGGATTTAATACC 49 10477 10496 586 544187 1546 1565GACAGTGACTTTAAGATAAA 27 10518 10537 587 544188 1572 1591TGTGATTGTATGTTTAATCT 30 10544 10563 588 544189 1578 1597AGGTTATGTGATTGTATGTT 35 10550 10569 589 544190 1583 1602CTTTAAGGTTATGTGATTGT 50 10555 10574 590 544191 1589 1608GGTATTCTTTAAGGTTATGT 54 10561 10580 591 544192 1656 1675ATTGATTCCCACATCACAAA 47 10628 10647 592 544193 1661 1680CTAAAATTGATTCCCACATC 69 10633 10652 593 544194 1665 1684CCATCTAAAATTGATTCCCA 74 10637 10656 594 544195 1771 1790TTGTGATATTAGCTCATATG 54 10743 10762 595 544196 1794 1813ACTAGTTTTTTAAACTGGGA 27 10766 10785 596 544197 1820 1839GTCAAGTTTAGAGTTTTAAC 18 10792 10811 597 544198 1826 1845TATTTAGTCAAGTTTAGAGT 12 10798 10817 598 544199 1907 1926TACACATACTCTGTGCTGAC 83 10879 10898 20 544200 1913 1932GATTTTTACACATACTCTGT 58 10885 10904 599 544201 2008 2027CTGCTTCATTAGGTTTCATA 62 10980 10999 600

TABLE 130 Inhibition of ANGPTL3 mRNA by 5-10-5 MOE gapmers targeting SEQID NO: 1 and 2 SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: NO: 2 NO: 2 ISISStart 1 Stop % Start Stop SEQ ID NO Site Site Sequence inhibition SiteSite NO 337520 N/A N/A CAGTGTTATTCAGATTGTAC 64 6517 6536 601 337521 N/AN/A AGTGTCTTACCATCATGTTT 40 6776 6795 602 337525 N/A N/ACACCAGCCTCCTAAAGGAGA 39 10212 10231 603 544292 N/A N/AGAGGAGGTGAAGTCAGTGAG 35 4815 4834 604 544293 N/A N/ATAGAGTAGAGGAGGTGAAGT 23 4822 4841 605 544294 N/A N/ATGTTTGATGTGTTTGAATAC 19 4863 4882 606 544295 N/A N/AGAAACAACAAGGGCAAAGGC 23 4898 4917 607 544296 N/A N/ATGTTTGATAACGACCCTAAG 43 4974 4993 608 544297 N/A N/ATTTTTGGTTAAGTGACCTTG 48 5016 5035 609 544298 N/A N/AGTAGAAGTTTTCAGGGATGG 23 5052 5071 610 544299 N/A N/AAGGAAGTAGAAGTTTTCAGG 5 5057 5076 611 544300 N/A N/A AGGTGAGTGTGCAGGAGAAA11 5085 5104 612 544301 N/A N/A TTAAATAAAGGTGAGTGTGC 14 5093 5112 613544302 N/A N/A AGTGCAGGAATAGAAGAGAT 35 5136 5155 614 544303 N/A N/ACATTTTAGTGCAGGAATAGA 21 5142 5161 615 544306 N/A N/ACTATATTCTGGAGTATATAC 39 5216 5235 616 544307 N/A N/ACAGTATTCTATATTCTGGAG 72 5223 5242 617 544308 N/A N/AGTGCCATACAGTATTCTATA 50 5231 5250 618 544309 N/A N/ACTGTGTGAATATGACATTAC 52 5281 5300 619 544310 N/A N/ATGAGGCACACTATTTCTAGT 47 5333 5352 620 544311 N/A N/AGACCTTTAATTATGAGGCAC 67 5345 5364 621 544312 N/A N/AGAATGTTGACCTTTAATTAT 23 5352 5371 622 544313 N/A N/ATTGTTGAATGTTGACCTTTA 69 5357 5376 623 544314 N/A N/ATCTACTAAGTAACTATGTGA 37 5915 5934 624 544315 N/A N/ACTCTTTTCTACTAAGTAACT 31 5921 5940 625 544316 N/A N/AAAGGATCTATTGTAAAGTTT 24 5956 5975 626 544317 N/A N/ACTAGGACCTTATTTAAAAGG 24 5972 5991 627 544318 N/A N/AATTTCCTAGGACCTTATTTA 8 5977 5996 628 544319 N/A N/A TTGACAGTAAGAAAAGCAGA28 6051 6070 629 544320 N/A N/A TTCTCATTGACAGTAAGAAA 56 6057 6076 630544321 N/A N/A AGTTTTTCTCATTGACAGTA 50 6062 6081 631 544322 N/A N/AATTGAATGATAGTTTTTCTC 42 6072 6091 632 544323 N/A N/ATTGGGTTTGCAATTTATTGA 36 6087 6106 633 544324 N/A N/AAGTGTGTTGGGTTTGCAATT 25 6093 6112 634 544325 N/A N/ATATTTAAGTGTGTTGGGTTT 27 6099 6118 635 544326 N/A N/AATATATTCAGTAGTTTATCG 25 6145 6164 636 544327 N/A N/AAGATGTTGGCAGGTTGGCAA 51 6184 6203 637 544328 N/A N/ATCTGTAGATGTTGGCAGGTT 48 6189 6208 638 544329 N/A N/ATTGATAATTTTTGACCTGTA 34 6215 6234 639 544330 N/A N/AGGCTTTCTTGATAATTTGAT 52 6230 6249 640 544331 N/A N/AGTCTTACTGATCTTCAGACC 27 6282 6301 641 544332 N/A N/ATTTAGGTCTTACTGATCTTC 14 6287 6306 642 544333 N/A N/ATCAGTTTTAGGTCTTACTGA 28 6292 6311 643 544334 N/A N/ATGATATTCTGTTCAGATTTT 44 6326 6345 644 544335 N/A N/ATAGAGACTGCTTTGCTTAGA 31 6388 6407 645 544336 N/A N/AAGGCCAAAAGTAGAGACTGC 29 6398 6417 646 544337 N/A N/AGGCAAAAAAGCAGACATTGG 38 6433 6452 647 544338 N/A N/AAATCAGGGACATTATTTAAT 13 6473 6492 648 544339 N/A N/ATATTTAATCAGGGACATTAT 28 6478 6497 649 544340 N/A N/ACTCAAAATATTTAATCAGGG 27 6485 6504 650 544341 N/A N/ATACCTGTTCTCAAAATATTT 18 6493 6512 651 544342 N/A N/AGTACAGATTACCTGTTCTCA 68 6501 6520 652 544343 N/A N/AGGTGTTTGATATTTAGATAA 25 6538 6557 653 544344 N/A N/ATTGTCTTTCAGTTCATAATG 29 6565 6584 654 544345 N/A N/AACAGTTTGTCTTTCAGTTCA 23 6570 6589 655 544346 N/A N/ATCTGAGCTGATAAAAGAATA 15 6657 6676 656 544347 N/A N/ACCCACCAAAGTGTCTTACCA 49 6784 6803 657 544348 N/A N/ACTTCAAGAAGGAAACCCACC 39 6798 6817 658 544349 N/A N/AAATAGCTTCAAGAAGGAAAC 12 6803 6822 659 544350 N/A N/AACAAGTCCTAAGAATAGGGA 25 6833 6852 660 544351 N/A N/AGTCTAGAACAAGTCCTAAGA 53 6840 6859 661 544352 N/A N/ATCTAATAATCAAGTCCATAT 33 6972 6991 662 544353 N/A N/AACCTTCTATATTATCTAATA 19 6985 7004 663 544354 N/A N/AGCATGTATCTCTTAAACAGG 50 7060 7079 664 544355 N/A N/ATTTCAGCATGTATCTCTTAA 79 7065 7084 21 544356 N/A N/A GTCCAGTGACCTTTAACTCC69 7092 7111 665 544357 N/A N/A TCTTACCAAACTATTTTCTT 28 7166 7185 666544358 N/A N/A GTAATGTTTATGTTAAAGCA 17 7226 7245 667 544359 N/A N/ATTGTGGCAAATGTAGCATTT 52 7251 7270 668 544360 N/A N/AGAGATTTCACTTGACATTTT 30 7277 7296 669 544361 N/A N/AGGAGCTTGAGATTTCACTTG 30 7284 7303 670 544362 N/A N/ACATCAGATTTAGTAATAGGA 0 7315 7334 671 544363 N/A N/A GTTATTACATCAGATTTAGT6 7322 7341 672 544365 N/A N/A CAGCAGGAATGCCTAGAATC 32 7350 7369 673544366 N/A N/A CTCCTTAGACAGGTTTTACC 31 7471 7490 674 544367 N/A N/AGTCTATTCTCCTTAGACAGG 23 7478 7497 675 544368 N/A N/AACCAGGTTAATCTTCCTAAT 71 7526 7545 22 544369 N/A N/A ATGAATGATTGAATGTAGTC26 7977 7996 676 544370 N/A N/A ATATGAAGGCTGAGACTGCT 58 8072 8091 677544371 N/A N/A ATAAATTATATGAAGGCTGA 7 8079 8098 678 544372 N/A N/AATATTTAAGAACAGACATGT 12 8175 8194 679 544373 N/A N/AAGTTATGATCATTGTAAGCC 60 8217 8236 23 544374 N/A N/A ATTTGTAACAGTTACTACTT51 8276 8295 680 544375 N/A N/A CACAGCTTATTTGTAACAGT 70 8284 8303 681544376 N/A N/A GGAGTGGTTCTTTTCACAGC 71 8298 8317 24 544377 N/A N/AGTGACTAATGCTAGGAGTGG 34 8311 8330 682 544378 N/A N/AGAATAGAGTGACTAATGCTA 45 8318 8337 683 544379 N/A N/AATGAGAGAATAGAGTGACTA 58 8324 8343 684 544380 N/A N/ATGGTCCTTTTAACTTCCAAT 70 8365 8384 25 544381 N/A N/A TATACTGTATGTCTGAGTTT66 8387 8406 685 544382 N/A N/A AACTAATTCATTATAAGCCA 67 8450 8469 686544383 N/A N/A GCATTGAGTTAACTAATTCA 64 8460 8479 26 544385 N/A N/ATTTGGATTTTAAACATCTGT 61 8528 8547 687 544386 N/A N/ATGTATGTGCTTTTTGGATTT 37 8539 8558 688 544387 N/A N/ACATGGATTTTTGTATGTGCT 62 8549 8568 689 544388 N/A N/ATCATTCATGGATTTTTGTAT 34 8554 8573 690 544389 N/A N/AACTTAGACATCATTCATGGA 55 8563 8582 691 544390 N/A N/AGTGAGTACTTAGACATCATT 66 8569 8588 692 544391 N/A N/ATTTATAAGTGAGTACTTAGA 36 8576 8595 693 544392 N/A N/AGTCTTCTACTTTATAAGTGA 65 8585 8604 694 544393 N/A N/AATGAATGTCTTCTACTTTAT 34 8591 8610 695 544394 N/A N/ACAAATAGTACTGAGCATTTA 30 8627 8646 696 544395 N/A N/ATTAGAAGATTTGGAGCTACA 54 8718 8737 697 544396 N/A N/ATCACTATTAGAAGATTTGGA 37 8724 8743 698 544397 N/A N/AGGGTTACACTCACTATTAGA 36 8733 8752 699 544398 N/A N/AACTTACCTGTCAGCCTTTTA 54 8758 8777 700 544399 N/A N/ACTTACCAGAATTAAGTGAGT 26 8785 8804 701 544400 N/A N/AAATACAAGTACAAATGGGTT 22 8810 8829 702 544401 N/A N/ACTGGTAAATACAAGTACAAA 55 8816 8835 703 544402 N/A N/AGGATTGCTGGTAAATACAAG 40 8822 8841 704 544403 N/A N/ATCATTTTAAGGATTGCTGGT 62 8831 8850 705 544404 N/A N/AAGTTAGTAGGAAGCTTCATT 56 8846 8865 706 544405 N/A N/AGCTATTGAGTTAGTAGGAAG 67 8853 8872 707 544407 N/A N/AAGCATGGTTCTTAATAACTT 67 9012 9031 708 544408 N/A N/ACTTTGTAGAAAAAGACAGGA 27 9062 9081 709 544409 N/A N/AACCTGGCCTTTGGTATTTGC 49 9096 9115 710 544410 N/A N/ACATCCATATACAGTCAAGAG 80 9174 9193 27 544411 N/A N/A AGTCTTTATATGGATAAACT15 9215 9234 711 544412 N/A N/A CGTCATTGGTAGAGGAATAT 51 9240 9259 712544413 N/A N/A GATTATCCTTTCTATAATGC 48 9321 9340 713 544414 N/A N/AGTCTTGAATCCCTTGATCAT 40 9436 9455 714 544415 N/A N/AGGTGCAACTAATTGAGTTGT 27 9459 9478 715 544416 N/A N/AGTGTTTTTTATTGGTGCAAC 31 9471 9490 716 544417 N/A N/AATTCTCCTGAAAAGAAAAGT 24 9544 9563 717 544418 N/A N/AATGCCACCACCAGCCTCCTA 73 10219 10238 718 544419 N/A N/AATATCCTTTAACAAATGGGT 62 11540 11559 719 544420 N/A N/AGCACTATATCCTTTAACAAA 50 11545 11564 720 544421 N/A N/AACTTGGGCACTATATCCTTT 68 11551 11570 721 544422 N/A N/AGAAACATGTCCTATGAGAGT 32 11918 11937 722 544424 N/A N/ATTGAGCACTTTAAGCAAAGT 7 12070 12089 723 544425 N/A N/AGGAATTTGAGCACTTTAAGC 34 12075 12094 724 544426 N/A N/ATAGATTAGACAACTGTGAGT 52 12101 12120 725 544427 N/A N/AAAAATGAAGGTCAAGTTTGA 17 12197 12216 726 544428 N/A N/AGTGAAAGCAAAATGAAGGTC 33 12205 12224 727 544429 N/A N/AGTATTGTGAAAGCAAAATGA 39 12210 12229 728 544430 N/A N/ATGGAGAGTATAGTATTGTGA 35 12221 12240 729 544438 N/A N/AAGGAATAGAAGAGATAAATA 10 5131 5150 730 544439 N/A N/ATGGAGTATATACAAATAATG 30 5208 5227 731 544440 N/A N/ATGTTTACATTGTAGATTAAT 15 5381 5400 732 544441 N/A N/ACAGAATATATAATATCTTGC 57 6035 6054 733 544442 N/A N/ATGCAATTTATTGAATGATAG 31 6080 6099 734 544443 N/A N/ACATAATACATAATTTGAACC 0 6251 6270 735 544444 N/A N/A ATAATTTTCAGTTTTAGGTC0 6299 6318 736 544445 N/A N/A TTTCAGTAATGTTTATGTTA 9 7231 7250 737544446 N/A N/A AATGCCTAGAATCAATAAAA 36 7343 7362 738 544447 N/A N/AGTAAATATTTGTAGATTAGC 49 8003 8022 739 544448 N/A N/AACAAATGTGTAATTGTTTGA 25 8101 8120 740 544449 N/A N/ATACTAACAAATGTGTAATTG 35 8106 8125 741 544450 N/A N/ATGATAAGTATATTTAAGAAC 35 8183 8202 742 544451 N/A N/ATTAACTTCCAATTAATTGAT 29 8357 8376 743 544452 N/A N/ATCTGTTATTTTATCTTGCTT 67 8513 8532 744 544453 N/A N/AATCACAATCCTTTTTATTAA 18 8921 8940 745 544454 N/A N/AAGAGACTTGAGTAATAATAA 25 9137 9156 746 544455 N/A N/AAACAAAATGAAACATGTCCT 59 11926 11945 747 544127 765 784CAGCAGGAATGCCATCATGT 33 N/A N/A 748 544128 819 838 TGATGGCATACATGCCACTT13 7404 7423 749 544129 828 847 TGCTGGGTCTGATGGCATAC 53 7413 7432 750544130 832 851 GAGTTGCTGGGTCTGATGGC 22 7417 7436 751 544131 841 860AAAACTTGAGAGTTGCTGGG 13 7426 7445 752 544132 848 867GACATGAAAAACTTGAGAGT 0 7433 7452 753 544133 859 878 ACATCACAGTAGACATGAAA27 7444 7463 754 233717 889 908 TGAATTAATGTCCATGGACT 58 7876 7895 14544134 915 934 AGTTTTGTGATCCATCTATT 46 7902 7921 755 544135 918 937TGAAGTTTTGTGATCCATCT 54 7905 7924 756 544136 926 945CGTTTCATTGAAGTTTTGTG 40 7913 7932 757 544137 946 965CCATATTTGTAGTTCTCCCA 45 7933 7952 758 544138 949 968AAACCATATTTGTAGTTCTC 41 7936 7955 759 544139 970 989AATTCTCCATCAAGCCTCCC 43 N/A N/A 760 233722 991 1010 ATCTTCTCTAGGCCCAACCA65 9566 9585 761 544432 997 1016 GAGTATATCTTCTCTAGGCC 40 9572 9591 762544140 1002 1021 CTATGGAGTATATCTTCTCT 28 9577 9596 763 544141 1008 1027GCTTCACTATGGAGTATATC 55 9583 9602 764 544142 1013 1032AGATTGCTTCACTATGGAGT 47 9588 9607 765 544143 1046 1065CCAGTCTTCCAACTCAATTC 33 9621 9640 766 544144 1052 1071GTCTTTCCAGTCTTCCAACT 59 9627 9646 767 544145 1055 1074GTTGTCTTTCCAGTCTTCCA 77 9630 9649 16 544146 1059 1078GTTTGTTGTCTTTCCAGTCT 58 9634 9653 768 544147 1062 1081AATGTTTGTTGTCTTTCCAG 43 9637 9656 769 544148 1095 1114CGTGATTTCCCAAGTAAAAA 57 9670 9689 770 544149 1160 1179GTTTTCCGGGATTGCATTGG 44 9735 9754 771 544150 1165 1184TCTTTGTTTTCCGGGATTGC 53 9740 9759 772 544151 1170 1189CCAAATCTTTGTTTTCCGGG 57 9745 9764 773 544152 1173 1192ACACCAAATCTTTGTTTTCC 44 9748 9767 774 544153 1178 1197AGAAAACACCAAATCTTTGT 36 9753 9772 775 544154 1183 1202CAAGTAGAAAACACCAAATC 29 9758 9777 776 544155 1188 1207GATCCCAAGTAGAAAACACC 29 9763 9782 777 544156 1195 1214GCTTTGTGATCCCAAGTAGA 71 9770 9789 17 544157 1198 1217TTTGCTTTGTGATCCCAAGT 66 9773 9792 778 544158 1202 1221TCCTTTTGCTTTGTGATCCC 53 9777 9796 779 544159 1208 1227GAAGTGTCCTTTTGCTTTGT 10 9783 9802 780 544160 1246 1265TGCCACCACCAGCCTCCTGA 65 N/A N/A 781 544161 1253 1272CTCATCATGCCACCACCAGC 59 10225 10244 782 544162 1269 1288GGTTGTTTTCTCCACACTCA 74 10241 10260 18 544163 1276 1295CCATTTAGGTTGTTTTCTCC 38 10248 10267 783 544164 1283 1302ATATTTACCATTTAGGTTGT 13 10255 10274 784 544165 1294 1313CTTGGTTTGTTATATTTACC 53 10266 10285 785 544166 1353 1372ACCTTCCATTTTGAGACTTC 70 10325 10344 19 544167 1363 1382ATAGAGTATAACCTTCCATT 69 10335 10354 786 544168 1367 1386TTTTATAGAGTATAACCTTC 34 10339 10358 787 544169 1374 1393TGGTTGATTTTATAGAGTAT 38 10346 10365 788 544170 1378 1397ATTTTGGTTGATTTTATAGA 0 10350 10369 789 544171 1383 1402TCAACATTTTGGTTGATTTT 12 10355 10374 790 544172 1390 1409GGATGGATCAACATTTTGGT 58 10362 10381 791 544173 1393 1412GTTGGATGGATCAACATTTT 66 10365 10384 792 544174 1396 1415TCTGTTGGATGGATCAACAT 49 10368 10387 793 544175 1401 1420CTGAATCTGTTGGATGGATC 60 10373 10392 794 544176 1407 1426AGCTTTCTGAATCTGTTGGA 64 10379 10398 795 544177 1414 1433CATTCAAAGCTTTCTGAATC 21 10386 10405 796 544178 1417 1436GTTCATTCAAAGCTTTCTGA 60 10389 10408 797 544179 1420 1439TCAGTTCATTCAAAGCTTTC 18 10392 10411 798 544180 1423 1442GCCTCAGTTCATTCAAAGCT 72 10395 10414 799 544181 1427 1446ATTTGCCTCAGTTCATTCAA 51 10399 10418 800 544182 1431 1450TTAAATTTGCCTCAGTTCAT 48 10403 10422 801 544183 1436 1455GCCTTTTAAATTTGCCTCAG 70 10408 10427 802 544184 1498 1517AGGATTTAATACCAGATTAT 44 10470 10489 803 544185 1502 1521CTTAAGGATTTAATACCAGA 47 10474 10493 804 544186 1505 1524TCTCTTAAGGATTTAATACC 44 10477 10496 805 544187 1546 1565GACAGTGACTTTAAGATAAA 38 10518 10537 806 544188 1572 1591TGTGATTGTATGTTTAATCT 47 10544 10563 807 544189 1578 1597AGGTTATGTGATTGTATGTT 43 10550 10569 808 544190 1583 1602CTTTAAGGTTATGTGATTGT 42 10555 10574 809 544191 1589 1608GGTATTCTTTAAGGTTATGT 60 10561 10580 810 544192 1656 1675ATTGATTCCCACATCACAAA 46 10628 10647 811 544193 1661 1680CTAAAATTGATTCCCACATC 65 10633 10652 812 544194 1665 1684CCATCTAAAATTGATTCCCA 70 10637 10656 813 544195 1771 1790TTGTGATATTAGCTCATATG 56 10743 10762 814 544196 1794 1813ACTAGTTTTTTAAACTGGGA 33 10766 10785 815 544197 1820 1839GTCAAGTTTAGAGTTTTAAC 39 10792 10811 816 544198 1826 1845TATTTAGTCAAGTTTAGAGT 21 10798 10817 817 544199 1907 1926TACACATACTCTGTGCTGAC 80 10879 10898 20 544200 1913 1932GATTTTTACACATACTCTGT 56 10885 10904 818 544201 2008 2027CTGCTTCATTAGGTTTCATA 65 10980 10999 819

TABLE 131 Inhibition of ANGPTL3 mRNA by 5-10-5 MOE gapmers targeting SEQID NO: 1 and 2 SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: NO: 2 NO: 2 ISISStart 1 Stop % Start Stop SEQ NO Site Site Sequence inhibition Site SiteID NO 337525 N/A N/A CACCAGCCTCCTAAAGGAGA 58 10212 10231 820 544204 N/AN/A GACTTCTTAACTCTATATAT 67 3076 3095 821 544205 N/A N/ACTAGACTTCTTAACTCTATA 61 3079 3098 822 544206 N/A N/AGACCTAGACTTCTTAACTCT 54 3082 3101 823 544207 N/A N/AGGAAGCAGACCTAGACTTCT 58 3089 3108 824 544208 N/A N/ATCTGGAAGCAGACCTAGACT 48 3092 3111 825 544209 N/A N/ATCTTCTGGAAGCAGACCTAG 54 3095 3114 826 544210 N/A N/ACTAATCTTTAGGGATTTAGG 57 11433 11452 827 544211 N/A N/ATGTATCTAATCTTTAGGGAT 53 11438 11457 828 544213 N/A N/ATAACTTGGGCACTATATCCT 74 11553 11572 829 544214 N/A N/AATTGACAAAGGTAGGTCACC 79 11576 11595 830 544215 N/A N/AATATGACATGTATATTGGAT 66 11620 11639 831 544216 N/A N/ATTTTGTACTTTTCTGGAACA 61 11704 11723 832 544217 N/A N/ATAGTCTGTGGTCCTGAAAAT 56 11748 11767 833 544218 N/A N/AAGCTTAGTCTGTGGTCCTGA 72 11752 11771 834 544219 N/A N/AGACAGCTTAGTCTGTGGTCC 74 11755 11774 835 544220 N/A N/AGTATTCTGGCCCTAAAAAAA 52 11789 11808 836 544221 N/A N/AATTTTGGTATTCTGGCCCTA 56 11795 11814 837 544222 N/A N/AGAAATTGTCCAATTTTTGGG 56 N/A N/A 838 544223 N/A N/A TTTGCATTTGAAATTGTCCA61 11837 11856 839 544224 N/A N/A GGAAGCAACTCATATATTAA 57 11869 11888840 544225 N/A N/A TATCAGAAAAAGATACCTGA 56 9821 9840 841 544226 N/A N/AATAATAGCTAATAATGTGGG 59 9875 9894 842 544227 N/A N/ATGCAGATAATAGCTAATAAT 60 9880 9899 843 544228 N/A N/ATGTCATTGCAGATAATAGCT 79 9886 9905 844 544229 N/A N/ATAAAAGTTGTCATTGCAGAT 59 9893 9912 845 544230 N/A N/ACGGATTTTTAAAAGTTGTCA 61 9901 9920 846 544231 N/A N/AGGGATTCGGATTTTTAAAAG 28 9907 9926 847 544232 N/A N/ATTTGGGATTCGGATTTTTAA 44 9910 9929 848 544233 N/A N/AACGCTTATTTGGGATTCGGA 72 9917 9936 849 544251 N/A N/ATTTAAGAGATTTACAAGTCA 52 2811 2830 850 544252 N/A N/AGACTACCTGTTTTTAAAAGC 48 2851 2870 851 544253 N/A N/ATATGGTGACTACCTGTTTTT 39 2857 2876 852 544254 N/A N/AACTTTGCTGTATTATAAACT 35 2890 2909 853 544255 N/A N/AATTGTATTTAACTTTGCTGT 35 2900 2919 854 544256 N/A N/AGAGCAACTAACTTAATAGGT 42 2928 2947 855 544257 N/A N/AGAAATGAGCAACTAACTTAA 32 2933 2952 856 544258 N/A N/AAATCAAAGAAATGAGCAACT 42 2940 2959 857 544259 N/A N/AACCTTCTTCCACATTGAGTT 44 2977 2996 858 544260 N/A N/ACACGAATGTAACCTTCTTCC 52 2987 3006 859 544261 N/A N/ATTAACTTGCACGAATGTAAC 45 2995 3014 860 544262 N/A N/ATATATATACCAATATTTGCC 43 3063 3082 861 544263 N/A N/ATCTTAACTCTATATATACCA 49 3072 3091 862 544264 N/A N/ACTTTAAGTGAAGTTACTTCT 53 3632 3651 863 544265 N/A N/ATCTACTTACTTTAAGTGAAG 44 3640 3659 864 544266 N/A N/AGAACCCTCTTTATTTTCTAC 46 3655 3674 865 544267 N/A N/AACATAAACATGAACCCTCTT 50 3665 3684 866 544268 N/A N/ACCACATTGAAAACATAAACA 57 3676 3695 867 544269 N/A N/AGCATGCCTTAGAAATATTTT 23 3707 3726 868 544270 N/A N/ACAATGCAACAAAGTATTTCA 37 3731 3750 869 544271 N/A N/ACTGGAGATTATTTTTCTTGG 61 3768 3787 870 544272 N/A N/ATTCATATATAACATTAGGGA 14 3830 3849 871 544273 N/A N/ATCAGTGTTTTCATATATAAC 32 3838 3857 872 544274 N/A N/AGACATAGTGTTCTAGATTGT 47 3900 3919 873 544275 N/A N/ACAATAGTGTAATGACATAGT 39 3912 3931 874 544276 N/A N/ATTACTTACCTTCAGTAATTT 35 3933 3952 875 544277 N/A N/AATCTTTTCCATTTACTGTAT 39 4005 4024 876 544278 N/A N/AAGAAAAAGCCCAGCATATTT 23 4037 4056 877 544279 N/A N/AGTATGCTTCTTTCAAATAGC 46 4130 4149 878 544280 N/A N/ACCTTCCCCTTGTATGCTTCT 47 4140 4159 879 544281 N/A N/ACCTGTAACACTATCATAATC 49 4207 4226 880 544282 N/A N/ATGACTTACCTGATTTTCTAT 24 4384 4403 881 544283 N/A N/AGATGGGACATACCATTAAAA 41 4407 4426 882 544284 N/A N/AGTGAAAGATGGGACATACCA 54 4413 4432 883 544285 N/A N/ACCTGTGTGAAAGATGGGACA 27 4418 4437 884 544286 N/A N/ACATTGGCTGCTATGAATTAA 45 4681 4700 885 544287 N/A N/AGATGACATTGGCTGCTATGA 49 4686 4705 886 544288 N/A N/AGAGAAACATGATCTAATTTG 33 4717 4736 887 544289 N/A N/AATGGAAAGCTATTGTGTGGT 42 4747 4766 888 544290 N/A N/AGTCTAAAGAGCCAATATGAG 39 4771 4790 889 544291 N/A N/AAATCTTGGTCTAAAGAGCCA 65 4778 4797 890 544361 N/A N/AGGAGCTTGAGATTTCACTTG 66 7284 7303 891 544362 N/A N/ACATCAGATTTAGTAATAGGA 61 7315 7334 892 544363 N/A N/AGTTATTACATCAGATTTAGT 63 7322 7341 893 544365 N/A N/ACAGCAGGAATGCCTAGAATC 72 7350 7369 894 544366 N/A N/ACTCCTTAGACAGGTTTTACC 67 7471 7490 895 544367 N/A N/AGTCTATTCTCCTTAGACAGG 59 7478 7497 896 544368 N/A N/AACCAGGTTAATCTTCCTAAT 79 7526 7545 22 544369 N/A N/A ATGAATGATTGAATGTAGTC56 7977 7996 897 544370 N/A N/A ATATGAAGGCTGAGACTGCT 73 8072 8091 898544371 N/A N/A ATAAATTATATGAAGGCTGA 51 8079 8098 899 544372 N/A N/AATATTTAAGAACAGACATGT 54 8175 8194 900 544373 N/A N/AAGTTATGATCATTGTAAGCC 77 8217 8236 23 544374 N/A N/A ATTTGTAACAGTTACTACTT69 8276 8295 901 544375 N/A N/A CACAGCTTATTTGTAACAGT 72 8284 8303 902544376 N/A N/A GGAGTGGTTCTTTTCACAGC 82 8298 8317 24 544377 N/A N/AGTGACTAATGCTAGGAGTGG 54 8311 8330 903 544378 N/A N/AGAATAGAGTGACTAATGCTA 55 8318 8337 904 544379 N/A N/AATGAGAGAATAGAGTGACTA 66 8324 8343 905 544380 N/A N/ATGGTCCTTTTAACTTCCAAT 79 8365 8384 25 544381 N/A N/A TATACTGTATGTCTGAGTTT72 8387 8406 906 544382 N/A N/A AACTAATTCATTATAAGCCA 56 8450 8469 907544383 N/A N/A GCATTGAGTTAACTAATTCA 78 8460 8479 26 544385 N/A N/ATTTGGATTTTAAACATCTGT 73 8528 8547 908 544386 N/A N/ATGTATGTGCTTTTTGGATTT 57 8539 8558 909 544387 N/A N/ACATGGATTTTTGTATGTGCT 64 8549 8568 910 544388 N/A N/ATCATTCATGGATTTTTGTAT 53 8554 8573 911 544389 N/A N/AACTTAGACATCATTCATGGA 66 8563 8582 912 544390 N/A N/AGTGAGTACTTAGACATCATT 74 8569 8588 913 544391 N/A N/ATTTATAAGTGAGTACTTAGA 32 8576 8595 914 544392 N/A N/AGTCTTCTACTTTATAAGTGA 63 8585 8604 915 544393 N/A N/AATGAATGTCTTCTACTTTAT 68 8591 8610 916 544394 N/A N/ACAAATAGTACTGAGCATTTA 53 8627 8646 917 544395 N/A N/ATTAGAAGATTTGGAGCTACA 55 8718 8737 918 544396 N/A N/ATCACTATTAGAAGATTTGGA 60 8724 8743 919 544397 N/A N/AGGGTTACACTCACTATTAGA 52 8733 8752 920 544398 N/A N/AACTTACCTGTCAGCCTTTTA 61 8758 8777 921 544399 N/A N/ACTTACCAGAATTAAGTGAGT 43 8785 8804 922 544400 N/A N/AAATACAAGTACAAATGGGTT 29 8810 8829 923 544401 N/A N/ACTGGTAAATACAAGTACAAA 76 8816 8835 924 544402 N/A N/AGGATTGCTGGTAAATACAAG 59 8822 8841 925 544403 N/A N/ATCATTTTAAGGATTGCTGGT 63 8831 8850 926 544404 N/A N/AAGTTAGTAGGAAGCTTCATT 54 8846 8865 927 544405 N/A N/AGCTATTGAGTTAGTAGGAAG 63 8853 8872 928 544407 N/A N/AAGCATGGTTCTTAATAACTT 69 9012 9031 929 544408 N/A N/ACTTTGTAGAAAAAGACAGGA 45 9062 9081 930 544409 N/A N/AACCTGGCCTTTGGTATTTGC 66 9096 9115 931 544410 N/A N/ACATCCATATACAGTCAAGAG 78 9174 9193 27 544411 N/A N/A AGTCTTTATATGGATAAACT46 9215 9234 932 544412 N/A N/A CGTCATTGGTAGAGGAATAT 45 9240 9259 933544413 N/A N/A GATTATCCTTTCTATAATGC 45 9321 9340 934 544414 N/A N/AGTCTTGAATCCCTTGATCAT 61 9436 9455 935 544415 N/A N/AGGTGCAACTAATTGAGTTGT 49 9459 9478 936 544416 N/A N/AGTGTTTTTTATTGGTGCAAC 46 9471 9490 937 544417 N/A N/AATTCTCCTGAAAAGAAAAGT 50 9544 9563 938 544418 N/A N/AATGCCACCACCAGCCTCCTA 73 10219 10238 939 544419 N/A N/AATATCCTTTAACAAATGGGT 68 11540 11559 940 544420 N/A N/AGCACTATATCCTTTAACAAA 74 11545 11564 941 544421 N/A N/AACTTGGGCACTATATCCTTT 68 11551 11570 942 544422 N/A N/AGAAACATGTCCTATGAGAGT 56 11918 11937 943 544424 N/A N/ATTGAGCACTTTAAGCAAAGT 15 12070 12089 944 544425 N/A N/AGGAATTTGAGCACTTTAAGC 35 12075 12094 945 544426 N/A N/ATAGATTAGACAACTGTGAGT 54 12101 12120 946 544427 N/A N/AAAAATGAAGGTCAAGTTTGA 45 12197 12216 947 544428 N/A N/AGTGAAAGCAAAATGAAGGTC 55 12205 12224 948 544429 N/A N/AGTATTGTGAAAGCAAAATGA 54 12210 12229 949 544430 N/A N/ATGGAGAGTATAGTATTGTGA 53 12221 12240 950 544433 N/A N/AGAGATTTACAAGTCAAAAAT 41 2806 2825 951 544434 N/A N/AATTTAACTTTGCTGTATTAT 29 2895 2914 952 544435 N/A N/AATCAATGCTAAATGAAATCA 34 2955 2974 953 544436 N/A N/ATATTTTCTGGAGATTATTTT 24 3774 3793 954 544437 N/A N/AAAAATGAATATTGGCAATTC 34 4159 4178 955 544446 N/A N/AAATGCCTAGAATCAATAAAA 50 7343 7362 956 544447 N/A N/AGTAAATATTTGTAGATTAGC 38 8003 8022 957 544448 N/A N/AACAAATGTGTAATTGTTTGA 43 8101 8120 958 544449 N/A N/ATACTAACAAATGTGTAATTG 59 8106 8125 959 544450 N/A N/ATGATAAGTATATTTAAGAAC 45 8183 8202 960 544451 N/A N/ATTAACTTCCAATTAATTGAT 55 8357 8376 961 544452 N/A N/ATCTGTTATTTTATCTTGCTT 67 8513 8532 962 544453 N/A N/AATCACAATCCTTTTTATTAA 39 8921 8940 963 544454 N/A N/AAGAGACTTGAGTAATAATAA 43 9137 9156 964 544455 N/A N/AAACAAAATGAAACATGTCCT 47 11926 11945 965 544059 23 42GATTTTCAATTTCAAGCAAC 74 3127 3146 966 337459 49 68 AGCTTAATTGTGAACATTTT77 3153 3172 967 544060 54 73 GAAGGAGCTTAATTGTGAAC 59 3158 3177 968544061 63 82 CAATAAAAAGAAGGAGCTTA 64 3167 3186 969 544062 66 85GAACAATAAAAAGAAGGAGC 67 3170 3189 970 544063 85 104 CTGGAGGAAATAACTAGAGG49 3189 3208 971 337460 88 107 ATTCTGGAGGAAATAACTAG 65 3192 3211 972544064 112 131 TCAAATGATGAATTGTCTTG 58 3216 3235 973 544065 138 157TTGATTTTGGCTCTGGAGAT 67 3242 3261 974 544066 145 164GCAAATCTTGATTTTGGCTC 82 3249 3268 975 233676 148 167ATAGCAAATCTTGATTTTGG 81 3252 3271 976 544067 156 175CGTCTAACATAGCAAATCTT 87 3260 3279 977 544068 174 193TGGCTAAAATTTTTACATCG 66 3278 3297 978 544069 178 197CCATTGGCTAAAATTTTTAC 41 3282 3301 979 544070 184 203AGGAGGCCATTGGCTAAAAT 36 3288 3307 980 544071 187 206TGAAGGAGGCCATTGGCTAA 44 3291 3310 981 544072 195 214GTCCCAACTGAAGGAGGCCA 59 3299 3318 982 544073 199 218CCATGTCCCAACTGAAGGAG 54 3303 3322 983 544074 202 221AGACCATGTCCCAACTGAAG 68 3306 3325 984 544075 206 225TTTAAGACCATGTCCCAACT 51 3310 3329 985 544076 209 228GTCTTTAAGACCATGTCCCA 64 3313 3332 986 544077 216 235GGACAAAGTCTTTAAGACCA 45 3320 3339 987 544078 222 241TCTTATGGACAAAGTCTTTA 40 3326 3345 988 544079 245 264TATGTCATTAATTTGGCCCT 30 3349 3368 989 544080 270 289GATCAAATATGTTGAGTTTT 65 3374 3393 990 233690 274 293GACTGATCAAATATGTTGAG 75 3378 3397 991 544081 316 335TCTTCTTTGATTTCACTGGT 86 3420 3439 992 544082 334 353CTTCTCAGTTCCTTTTCTTC 69 3438 3457 993 544083 337 356GTTCTTCTCAGTTCCTTTTC 77 3441 3460 994 544084 341 360TGTAGTTCTTCTCAGTTCCT 75 3445 3464 995 544431 345 364TATATGTAGTTCTTCTCAGT 15 3449 3468 996 544086 348 367GTTTATATGTAGTTCTTCTC 65 3452 3471 997 544087 352 371TGTAGTTTATATGTAGTTCT 49 3456 3475 998 544088 356 375GACTTGTAGTTTATATGTAG 21 3460 3479 999 544089 364 383TCATTTTTGACTTGTAGTTT 60 3468 3487 1000 544090 369 388CCTCTTCATTTTTGACTTGT 83 3473 3492 1001 544091 375 394TCTTTACCTCTTCATTTTTG 75 3479 3498 1002 544092 380 399CATATTCTTTACCTCTTCAT 77 3484 3503 1003 544093 384 403GTGACATATTCTTTACCTCT 76 3488 3507 1004 544094 392 411GAGTTCAAGTGACATATTCT 71 3496 3515 1005 544095 398 417TGAGTTGAGTTCAAGTGACA 44 3502 3521 1006 544096 403 422AGTTTTGAGTTGAGTTCAAG 33 3507 3526 1007 544097 406 425TCAAGTTTTGAGTTGAGTTC 69 3510 3529 1008 544098 414 433GGAGGCTTTCAAGTTTTGAG 68 3518 3537 1009 544099 423 442TTTCTTCTAGGAGGCTTTCA 79 3527 3546 1010 544100 427 446ATTTTTTCTTCTAGGAGGCT 63 3531 3550 1011 544101 432 451GTAGAATTTTTTCTTCTAGG 56 3536 3555 1012 544102 462 481GCTCTTCTAAATATTTCACT 85 3566 3585 1013 544103 474 493AGTTAGTTAGTTGCTCTTCT 71 3578 3597 1014 544104 492 511CAGGTTGATTTTGAATTAAG 69 3596 3615 1015 544105 495 514TTTCAGGTTGATTTTGAATT 53 3599 3618 1016 544106 499 518GGAGTTTCAGGTTGATTTTG 64 3603 3622 1017 544107 504 523GTTCTGGAGTTTCAGGTTGA 74 3608 3627 1018 544108 526 545TTAAGTGAAGTTACTTCTGG 60 3630 3649 1019 544109 555 574TGCTATTATCTTGTTTTTCT 63 4293 4312 1020 544110 564 583GGTCTTTGATGCTATTATCT 65 4302 4321 1021 544111 567 586GAAGGTCTTTGATGCTATTA 49 4305 4324 1022 544112 572 591CTGGAGAAGGTCTTTGATGC 65 4310 4329 1023 544113 643 662CTGAGCTGATTTTCTATTTC 64 N/A N/A 1024 337477 664 683 GGTTCTTGAATACTAGTCCT82 6677 6696 234 544114 673 692 ATTTCTGTGGGTTCTTGAAT 57 6686 6705 1025337478 675 694 AAATTTCTGTGGGTTCTTGA 29 6688 6707 235 544115 678 697GAGAAATTTCTGTGGGTTCT 68 6691 6710 1026 544116 682 701GATAGAGAAATTTCTGTGGG 54 6695 6714 1027 544117 689 708CTTGGAAGATAGAGAAATTT 36 6702 6721 1028 337479 692 711TGGCTTGGAAGATAGAGAAA 54 6705 6724 236 544118 699 718GTGCTCTTGGCTTGGAAGAT 64 6712 6731 1029 544119 703 722CTTGGTGCTCTTGGCTTGGA 68 6716 6735 1030 544120 707 726AGTTCTTGGTGCTCTTGGCT 91 6720 6739 15 233710 710 729 AGTAGTTCTTGGTGCTCTTG80 6723 6742 233 544121 713 732 GGGAGTAGTTCTTGGTGCTC 76 6726 6745 1031544122 722 741 CTGAAGAAAGGGAGTAGTTC 55 6735 6754 1032 544123 752 771ATCATGTTTTACATTTCTTA 52 6765 6784 1033 544124 755 774GCCATCATGTTTTACATTTC 61 N/A N/A 1034 544125 759 778 GAATGCCATCATGTTTTACA30 N/A N/A 1035 544126 762 781 CAGGAATGCCATCATGTTTT 34 N/A N/A 1036337487 804 823 CACTTGTATGTTCACCTCTG 83 7389 7408 28 233717 889 908TGAATTAATGTCCATGGACT 75 7876 7895 14 544202 2081 2100AAAGTCAATGTGACTTAGTA 70 11053 11072 1037 544203 2104 2123AAGGTATAGTGATACCTCAT 84 11076 11095 1038

TABLE 132 Inhibition of ANGPTL3 mRNA by 5-10-5 MOE gapmers targeting SEQID NO: 1 and 2 SEQ SEQ ID SEQ SEQ ID NO: NO: ID NO: ID NO: ISIS 1 Start1 Stop % 2 Start 2 Stop SEQ NO Site Site Sequence inhibition Site SiteID NO 560535 N/A N/A ACTGTTTTCTTCTGGAAGCA 0 3102 3121 1039 560536 N/AN/A AAATAAGGTATAGTGATACC 0 11080 11099 1040 560537 N/A N/AACAAATAAGGTATAGTGATA 1 11082 11101 1041 560538 N/A N/ATAACAAATAAGGTATAGTGA 0 11084 11103 1042 560539 N/A N/ATTTAACAAATAAGGTATAGT 16 11086 11105 1043 560540 N/A N/AATATATTTTAACAAATAAGG 0 11092 11111 1044 560541 N/A N/ACAGTATATATTTTAACAAAT 0 11096 11115 1045 560542 N/A N/ATACAGTATATATTTTAACAA 0 11098 11117 1046 560543 N/A N/ATATACAGTATATATTTTAAC 0 11100 11119 1047 560544 N/A N/AATAGTATTAAGTGTTAAAAT 0 11130 11149 1048 560545 N/A N/ATCATAGTATTAAGTGTTAAA 0 11132 11151 1049 560546 N/A N/AGTTTTCATAGTATTAAGTGT 26 11136 11155 1050 560547 N/A N/AATTATTTGTTTTCATAGTAT 0 11143 11162 1051 560548 N/A N/ACTTTACAATTATTTGTTTTC 0 11150 11169 1052 560549 N/A N/AATTCCTTTACAATTATTTGT 21 11154 11173 1053 560550 N/A N/AAGATTCCTTTACAATTATTT 18 11156 11175 1054 560551 N/A N/ACAAGATTCCTTTACAATTAT 21 11158 11177 1055 560552 N/A N/AGACAAGATTCCTTTACAATT 55 11160 11179 1056 560553 N/A N/ACTGACAAGATTCCTTTACAA 47 11162 11181 1057 560554 N/A N/AAATCTGACAAGATTCCTTTA 52 11165 11184 1058 560555 N/A N/AGTAATCTGACAAGATTCCTT 56 11167 11186 1059 560556 N/A N/ACTGTAATCTGACAAGATTCC 51 11169 11188 1060 560557 N/A N/ATACTGTAATCTGACAAGATT 18 11171 11190 1061 560558 N/A N/ACTTACTGTAATCTGACAAGA 33 11173 11192 1062 560559 N/A N/ATTCTTACTGTAATCTGACAA 47 11175 11194 1063 560560 N/A N/ACATTCTTACTGTAATCTGAC 65 11177 11196 1064 560561 N/A N/ATTCATTCTTACTGTAATCTG 54 11179 11198 1065 560562 N/A N/ATGTTCATTCTTACTGTAATC 44 11181 11200 1066 560563 N/A N/ATATGTTCATTCTTACTGTAA 39 11183 11202 1067 560564 N/A N/AAATATGTTCATTCTTACTGT 0 11185 11204 1068 560565 N/A N/AACAAATATGTTCATTCTTAC 3 11188 11207 1069 560566 N/A N/ACCACAAATATGTTCATTCTT 75 11190 11209 42 560567 N/A N/ATGCCACAAATATGTTCATTC 80 11192 11211 43 560568 N/A N/ACGATGCCACAAATATGTTCA 64 11195 11214 1070 560569 N/A N/ACTCGATGCCACAAATATGTT 65 11197 11216 1071 560570 N/A N/AAACTCGATGCCACAAATATG 46 11199 11218 1072 560571 N/A N/ATTAACTCGATGCCACAAATA 52 11201 11220 1073 560572 N/A N/ACTTTAACTCGATGCCACAAA 66 11203 11222 1074 560573 N/A N/AAACTTTAACTCGATGCCACA 53 11205 11224 1075 560574 N/A N/ATAAACTTTAACTCGATGCCA 72 11207 11226 44 560575 N/A N/AAATATAAACTTTAACTCGAT 6 11211 11230 1076 560576 N/A N/AGAAATATAAACTTTAACTCG 17 11213 11232 1077 560577 N/A N/AGGGAAATATAAACTTTAACT 0 11215 11234 1078 560578 N/A N/AGAATCACAGCATATTTAGGG 46 11233 11252 1079 560579 N/A N/ATAGAATCACAGCATATTTAG 32 11235 11254 1080 560580 N/A N/AGTATTAGAATCACAGCATAT 51 11239 11258 1081 560581 N/A N/AATGTATTAGAATCACAGCAT 64 11241 11260 1082 560582 N/A N/AGAATGTATTAGAATCACAGC 44 11243 11262 1083 560583 N/A N/AACGAATGTATTAGAATCACA 44 11245 11264 1084 560584 N/A N/AACACGAATGTATTAGAATCA 41 11247 11266 1085 560585 N/A N/ACTACACGAATGTATTAGAAT 15 11249 11268 1086 560586 N/A N/AACCTACACGAATGTATTAGA 37 11251 11270 1087 560587 N/A N/AAAACCTACACGAATGTATTA 3 11253 11272 1088 560588 N/A N/AGAAAACCTACACGAATGTAT 27 11255 11274 1089 560589 N/A N/ATTGAAAACCTACACGAATGT 19 11257 11276 1090 560590 N/A N/AACTTGAAAACCTACACGAAT 21 11259 11278 1091 560591 N/A N/ACTACTTGAAAACCTACACGA 43 11261 11280 1092 560592 N/A N/ATATTTCTACTTGAAAACCTA 29 11266 11285 1093 560593 N/A N/ATTTATTTCTACTTGAAAACC 2 11268 11287 1094 560594 N/A N/AGGTTTATTTCTACTTGAAAA 27 11270 11289 1095 560595 N/A N/AGAGGTTTATTTCTACTTGAA 45 11272 11291 1096 560596 N/A N/AACGAGGTTTATTTCTACTTG 75 11274 11293 45 560597 N/A N/ATTACGAGGTTTATTTCTACT 49 11276 11295 1097 560598 N/A N/ATGTTACGAGGTTTATTTCTA 39 11278 11297 1098 560599 N/A N/ACTTGTTACGAGGTTTATTTC 32 11280 11299 1099 560600 N/A N/AAACTTGTTACGAGGTTTATT 27 11282 11301 1100 560601 N/A N/AGTAACTTGTTACGAGGTTTA 55 11284 11303 1101 560602 N/A N/ACAGTAACTTGTTACGAGGTT 51 11286 11305 1102 560603 N/A N/ATTCAGTAACTTGTTACGAGG 40 11288 11307 1103 560604 N/A N/ACGTTCAGTAACTTGTTACGA 53 11290 11309 1104 560605 N/A N/ACTTGTCAGGCTGTTTAAACG 24 11308 11327 1105 560606 N/A N/ATGCTTGTCAGGCTGTTTAAA 46 11310 11329 1106 560607 N/A N/ACATGCTTGTCAGGCTGTTTA 72 11312 11331 46 560608 N/A N/ATACATGCTTGTCAGGCTGTT 72 11314 11333 47 560609 N/A N/ATATACATGCTTGTCAGGCTG 63 11316 11335 1107 560610 N/A N/ATATATACATGCTTGTCAGGC 55 11318 11337 1108 560611 N/A N/ACATATATACATGCTTGTCAG 47 11320 11339 1109 560235 2 21TGGAACTGTTTTCTTCTGGA 43 3106 3125 1110 337526 4 23 CGTGGAACTGTTTTCTTCTG54 3108 3127 1111 560236 25 44 TTGATTTTCAATTTCAAGCA 91 3129 3148 30560237 27 46 TCTTGATTTTCAATTTCAAG 33 3131 3150 1112 560238 32 51TTTTATCTTGATTTTCAATT 0 3136 3155 1113 560239 35 54 CATTTTTATCTTGATTTTCA6 3139 3158 1114 560240 43 62 ATTGTGAACATTTTTATCTT 0 3147 3166 1115560241 45 64 TAATTGTGAACATTTTTATC 20 3149 3168 1116 560242 56 75AAGAAGGAGCTTAATTGTGA 39 3160 3179 1117 560243 58 77 AAAAGAAGGAGCTTAATTGT17 3162 3181 1118 560244 60 79 TAAAAAGAAGGAGCTTAATT 0 3164 3183 1119560245 75 94 TAACTAGAGGAACAATAAAA 37 3179 3198 1120 560246 77 96AATAACTAGAGGAACAATAA 3 3181 3200 1121 560247 79 98 GAAATAACTAGAGGAACAAT13 3183 3202 1122 560248 81 100 AGGAAATAACTAGAGGAACA 28 3185 3204 1123560249 83 102 GGAGGAAATAACTAGAGGAA 12 3187 3206 1124 560250 90 109CAATTCTGGAGGAAATAACT 34 3194 3213 1125 560251 92 111ATCAATTCTGGAGGAAATAA 32 3196 3215 1126 560252 96 115CTTGATCAATTCTGGAGGAA 15 3200 3219 1127 560253 98 117GTCTTGATCAATTCTGGAGG 53 3202 3221 1128 560254 100 119TTGTCTTGATCAATTCTGGA 48 3204 3223 1129 560255 102 121AATTGTCTTGATCAATTCTG 23 3206 3225 1130 560256 104 123TGAATTGTCTTGATCAATTC 14 3208 3227 1131 560257 106 125GATGAATTGTCTTGATCAAT 46 3210 3229 1132 560258 108 127ATGATGAATTGTCTTGATCA 33 3212 3231 1133 560259 110 129AAATGATGAATTGTCTTGAT 24 3214 3233 1134 560260 114 133AATCAAATGATGAATTGTCT 25 3218 3237 1135 560261 116 135AGAATCAAATGATGAATTGT 16 3220 3239 1136 560262 119 138TAGAGAATCAAATGATGAAT 7 3223 3242 1137 560263 126 145CTGGAGATAGAGAATCAAAT 40 3230 3249 1138 560264 128 147CTCTGGAGATAGAGAATCAA 51 3232 3251 1139 560265 130 149GGCTCTGGAGATAGAGAATC 63 3234 3253 31 560266 132 151 TTGGCTCTGGAGATAGAGAA49 3236 3255 1140 560267 135 154 ATTTTGGCTCTGGAGATAGA 49 3239 3258 1141560268 140 159 TCTTGATTTTGGCTCTGGAG 69 3244 3263 32 560269 142 161AATCTTGATTTTGGCTCTGG 53 3246 3265 1142 560270 150 169ACATAGCAAATCTTGATTTT 25 3254 3273 1143 560271 152 171TAACATAGCAAATCTTGATT 0 3256 3275 1144 560272 154 173TCTAACATAGCAAATCTTGA 53 3258 3277 1145 560273 176 195ATTGGCTAAAATTTTTACAT 12 3280 3299 1146 560274 180 199GGCCATTGGCTAAAATTTTT 34 3284 3303 1147 560275 182 201GAGGCCATTGGCTAAAATTT 26 3286 3305 1148 560276 189 208ACTGAAGGAGGCCATTGGCT 51 3293 3312 1149 560277 191 210CAACTGAAGGAGGCCATTGG 28 3295 3314 1150 560278 193 212CCCAACTGAAGGAGGCCATT 10 3297 3316 1151 560279 197 216ATGTCCCAACTGAAGGAGGC 0 3301 3320 1152 560280 204 223TAAGACCATGTCCCAACTGA 13 3308 3327 1153 560281 211 230AAGTCTTTAAGACCATGTCC 4 3315 3334 1154 560282 213 232CAAAGTCTTTAAGACCATGT 24 3317 3336 1155 560283 219 238TATGGACAAAGTCTTTAAGA 8 3323 3342 1156 560284 224 243CGTCTTATGGACAAAGTCTT 11 3328 3347 1157 560285 242 261GTCATTAATTTGGCCCTTCG 57 3346 3365 33 560286 247 266 AATATGTCATTAATTTGGCC0 3351 3370 1158 560287 249 268 GAAATATGTCATTAATTTGG 0 3353 3372 1159560288 252 271 TTTGAAATATGTCATTAATT 4 3356 3375 1160 560289 256 275AGTTTTTGAAATATGTCATT 7 3360 3379 1161 560290 258 277TGAGTTTTTGAAATATGTCA 41 3362 3381 1162 560291 267 286CAAATATGTTGAGTTTTTGA 30 3371 3390 1163 560292 272 291CTGATCAAATATGTTGAGTT 32 3376 3395 1164 560293 276 295AAGACTGATCAAATATGTTG 37 3380 3399 1165 560294 280 299TAAAAAGACTGATCAAATAT 0 3384 3403 1166 560295 282 301CATAAAAAGACTGATCAAAT 6 3386 3405 1167 560296 284 303ATCATAAAAAGACTGATCAA 10 3388 3407 1168 560297 287 306TAGATCATAAAAAGACTGAT 0 3391 3410 1169 560298 289 308GATAGATCATAAAAAGACTG 21 3393 3412 1170 560299 291 310GCGATAGATCATAAAAAGAC 20 3395 3414 1171 560300 293 312CAGCGATAGATCATAAAAAG 16 3397 3416 1172 560301 295 314TGCAGCGATAGATCATAAAA 38 3399 3418 1173 560302 297 316TTTGCAGCGATAGATCATAA 32 3401 3420 1174 560303 299 318GGTTTGCAGCGATAGATCAT 34 3403 3422 1175 560304 301 320CTGGTTTGCAGCGATAGATC 25 3405 3424 1176 560305 303 322CACTGGTTTGCAGCGATAGA 28 3407 3426 1177 560306 305 324TTCACTGGTTTGCAGCGATA 65 3409 3428 34 560307 307 326 ATTTCACTGGTTTGCAGCGA23 3411 3430 1178 560308 310 329 TTGATTTCACTGGTTTGCAG 5 3414 3433 1179560309 318 337 CTTCTTCTTTGATTTCACTG 25 3422 3441 1180 560310 327 346GTTCCTTTTCTTCTTCTTTG 19 3431 3450 1181 544120 707 726AGTTCTTGGTGCTCTTGGCT 77 6720 6739 15 560311 801 820 TTGTATGTTCACCTCTGTTA25 7386 7405 1182 560312 802 821 CTTGTATGTTCACCTCTGTT 37 7387 7406 1183337487 804 823 CACTTGTATGTTCACCTCTG 83 7389 7408 28 560313 806 825GCCACTTGTATGTTCACCTC 40 7391 7410 1184 560314 807 826TGCCACTTGTATGTTCACCT 56 7392 7411 1185 560315 808 827ATGCCACTTGTATGTTCACC 39 7393 7412 1186 337488 809 828CATGCCACTTGTATGTTCAC 19 7394 7413 1187 560316 810 829ACATGCCACTTGTATGTTCA 26 7395 7414 1188 560317 811 830TACATGCCACTTGTATGTTC 20 7396 7415 1189 560318 814 833GCATACATGCCACTTGTATG 2 7399 7418 1190 560319 815 834GGCATACATGCCACTTGTAT 24 7400 7419 1191 560320 816 835TGGCATACATGCCACTTGTA 7 7401 7420 1192 560321 817 836ATGGCATACATGCCACTTGT 0 7402 7421 1193 560322 821 840TCTGATGGCATACATGCCAC 26 7406 7425 1194 560323 822 841GTCTGATGGCATACATGCCA 39 7407 7426 1195 560324 824 843GGGTCTGATGGCATACATGC 15 7409 7428 1196 560325 825 844TGGGTCTGATGGCATACATG 23 7410 7429 1197 560326 826 845CTGGGTCTGATGGCATACAT 9 7411 7430 1198 560327 834 853GAGAGTTGCTGGGTCTGATG 0 7419 7438 1199 560328 835 854TGAGAGTTGCTGGGTCTGAT 2 7420 7439 1200 560329 836 855TTGAGAGTTGCTGGGTCTGA 35 7421 7440 1201 560330 837 856CTTGAGAGTTGCTGGGTCTG 17 7422 7441 1202 560331 838 857ACTTGAGAGTTGCTGGGTCT 0 7423 7442 1203 560332 839 858AACTTGAGAGTTGCTGGGTC 13 7424 7443 1204 560333 843 862GAAAAACTTGAGAGTTGCTG 22 7428 7447 1205 560334 844 863TGAAAAACTTGAGAGTTGCT 16 7429 7448 1206 560335 845 864ATGAAAAACTTGAGAGTTGC 10 7430 7449 1207 560336 846 865CATGAAAAACTTGAGAGTTG 2 7431 7450 1208 560337 851 870GTAGACATGAAAAACTTGAG 13 7436 7455 1209 560338 853 872CAGTAGACATGAAAAACTTG 3 7438 7457 1210 560339 861 880TAACATCACAGTAGACATGA 30 7446 7465 1211 560340 862 881ATAACATCACAGTAGACATG 34 7447 7466 1212 560341 863 882TATAACATCACAGTAGACAT 0 7448 7467 1213 560342 864 883ATATAACATCACAGTAGACA 10 7449 7468 1214 560343 865 884GATATAACATCACAGTAGAC 9 7450 7469 1215 560344 866 885TGATATAACATCACAGTAGA 20 7451 7470 1216 337490 867 886CTGATATAACATCACAGTAG 24 7452 7471 1217 560345 868 887CCTGATATAACATCACAGTA 36 7453 7472 1218 560346 869 888ACCTGATATAACATCACAGT 35 7454 7473 1219 560347 870 889TACCTGATATAACATCACAG 26 7455 7474 1220 560348 871 890CTACCTGATATAACATCACA 38 N/A N/A 1221 560349 872 891 ACTACCTGATATAACATCAC12 N/A N/A 1222 560350 873 892 GACTACCTGATATAACATCA 28 N/A N/A 1223560351 874 893 GGACTACCTGATATAACATC 15 N/A N/A 1224 560352 875 894TGGACTACCTGATATAACAT 0 N/A N/A 1225 560353 876 895 ATGGACTACCTGATATAACA11 N/A N/A 1226 337491 877 896 CATGGACTACCTGATATAAC 3 N/A N/A 1227560354 878 897 CCATGGACTACCTGATATAA 0 N/A N/A 1228 560355 879 898TCCATGGACTACCTGATATA 13 N/A N/A 1229 560356 880 899 GTCCATGGACTACCTGATAT50 N/A N/A 1230 560357 881 900 TGTCCATGGACTACCTGATA 12 N/A N/A 1231560358 882 901 ATGTCCATGGACTACCTGAT 20 N/A N/A 1232 560359 883 902AATGTCCATGGACTACCTGA 16 7870 7889 1233 560360 884 903TAATGTCCATGGACTACCTG 26 7871 7890 1234 560361 885 904TTAATGTCCATGGACTACCT 31 7872 7891 1235 560362 886 905ATTAATGTCCATGGACTACC 42 7873 7892 1236 560363 887 906AATTAATGTCCATGGACTAC 21 7874 7893 1237 560364 891 910GTTGAATTAATGTCCATGGA 18 7878 7897 1238 560365 892 911TGTTGAATTAATGTCCATGG 36 7879 7898 1239 560366 893 912ATGTTGAATTAATGTCCATG 13 7880 7899 1240 560367 894 913GATGTTGAATTAATGTCCAT 14 7881 7900 1241 560368 895 914CGATGTTGAATTAATGTCCA 30 7882 7901 1242 560369 896 915TCGATGTTGAATTAATGTCC 29 7883 7902 1243 560370 897 916TTCGATGTTGAATTAATGTC 4 7884 7903 1244 560371 898 917ATTCGATGTTGAATTAATGT 22 7885 7904 1245 560372 899 918TATTCGATGTTGAATTAATG 0 7886 7905 1246 560373 900 919CTATTCGATGTTGAATTAAT 0 7887 7906 1247 337492 901 920TCTATTCGATGTTGAATTAA 59 7888 7907 29 560374 902 921 ATCTATTCGATGTTGAATTA18 7889 7908 1248 560375 903 922 CATCTATTCGATGTTGAATT 27 7890 7909 1249560376 904 923 CCATCTATTCGATGTTGAAT 40 7891 7910 1250 560377 905 924TCCATCTATTCGATGTTGAA 23 7892 7911 1251 560378 906 925ATCCATCTATTCGATGTTGA 47 7893 7912 1252 560379 907 926GATCCATCTATTCGATGTTG 46 7894 7913 1253 560380 908 927TGATCCATCTATTCGATGTT 16 7895 7914 1254 560381 909 928GTGATCCATCTATTCGATGT 24 7896 7915 1255 560382 910 929TGTGATCCATCTATTCGATG 21 7897 7916 1256 560383 911 930TTGTGATCCATCTATTCGAT 19 7898 7917 1257 560384 1273 1292TTTAGGTTGTTTTCTCCACA 35 10245 10264 1258 560385 1274 1293ATTTAGGTTGTTTTCTCCAC 34 10246 10265 1259 560386 1278 1297TACCATTTAGGTTGTTTTCT 15 10250 10269 1260 560387 1286 1305GTTATATTTACCATTTAGGT 20 10258 10277 1261 560388 1287 1306TGTTATATTTACCATTTAGG 17 10259 10278 1262 560389 1288 1307TTGTTATATTTACCATTTAG 21 10260 10279 1263 560390 1289 1308TTTGTTATATTTACCATTTA 4 10261 10280 1264 560391 1292 1311TGGTTTGTTATATTTACCAT 23 10264 10283 1265 560392 1296 1315CTCTTGGTTTGTTATATTTA 63 10268 10287 1266 560393 1297 1316GCTCTTGGTTTGTTATATTT 61 10269 10288 1267 560394 1298 1317TGCTCTTGGTTTGTTATATT 51 10270 10289 1268 560395 1301 1320TTTTGCTCTTGGTTTGTTAT 2 10273 10292 1269 560396 1302 1321ATTTTGCTCTTGGTTTGTTA 0 10274 10293 1270 560397 1303 1322GATTTTGCTCTTGGTTTGTT 0 10275 10294 1271 560398 1304 1323AGATTTTGCTCTTGGTTTGT 16 10276 10295 1272 560399 1305 1324TAGATTTTGCTCTTGGTTTG 28 10277 10296 1273 560400 1307 1326CTTAGATTTTGCTCTTGGTT 69 10279 10298 35 560401 1308 1327GCTTAGATTTTGCTCTTGGT 77 10280 10299 36 560402 1309 1328GGCTTAGATTTTGCTCTTGG 72 10281 10300 37 560403 1315 1334CTCTCTGGCTTAGATTTTGC 38 10287 10306 1274 560404 1316 1335CCTCTCTGGCTTAGATTTTG 49 10288 10307 1275 560405 1317 1336TCCTCTCTGGCTTAGATTTT 46 10289 10308 1276 560406 1321 1340CTTCTCCTCTCTGGCTTAGA 40 10293 10312 1277 560407 1322 1341TCTTCTCCTCTCTGGCTTAG 57 10294 10313 1278 560408 1323 1342CTCTTCTCCTCTCTGGCTTA 40 10295 10314 1279 337505 1328 1347TAATCCTCTTCTCCTCTCTG 28 10300 10319 1280 560409 1329 1348ATAATCCTCTTCTCCTCTCT 30 10301 10320 1281 560410 1330 1349GATAATCCTCTTCTCCTCTC 9 10302 10321 1282 560411 1331 1350AGATAATCCTCTTCTCCTCT 23 10303 10322 1283 560412 1332 1351AAGATAATCCTCTTCTCCTC 12 10304 10323 1284 560413 1333 1352CAAGATAATCCTCTTCTCCT 40 10305 10324 1285 560414 1334 1353CCAAGATAATCCTCTTCTCC 52 10306 10325 1286 560415 1335 1354TCCAAGATAATCCTCTTCTC 56 10307 10326 1287 560416 1336 1355TTCCAAGATAATCCTCTTCT 60 10308 10327 1288 560417 1337 1356CTTCCAAGATAATCCTCTTC 58 10309 10328 1289 560418 1338 1357ACTTCCAAGATAATCCTCTT 31 10310 10329 1290 560419 1339 1358GACTTCCAAGATAATCCTCT 52 10311 10330 1291 560420 1340 1359AGACTTCCAAGATAATCCTC 49 10312 10331 1292 560421 1341 1360GAGACTTCCAAGATAATCCT 56 10313 10332 1293 337506 1342 1361TGAGACTTCCAAGATAATCC 49 10314 10333 1294 560422 1343 1362TTGAGACTTCCAAGATAATC 34 10315 10334 1295 560423 1344 1363TTTGAGACTTCCAAGATAAT 14 10316 10335 1296 560424 1345 1364TTTTGAGACTTCCAAGATAA 27 10317 10336 1297 560425 1346 1365ATTTTGAGACTTCCAAGATA 23 10318 10337 1298 560426 1348 1367CCATTTTGAGACTTCCAAGA 40 10320 10339 1299 560427 1351 1370CTTCCATTTTGAGACTTCCA 58 10323 10342 1300 560428 1355 1374TAACCTTCCATTTTGAGACT 36 10327 10346 1301 560429 1356 1375ATAACCTTCCATTTTGAGAC 51 10328 10347 1302 560430 1357 1376TATAACCTTCCATTTTGAGA 33 10329 10348 1303 560431 1358 1377GTATAACCTTCCATTTTGAG 53 10330 10349 1304 337508 1360 1379GAGTATAACCTTCCATTTTG 28 10332 10351 1305 560432 1361 1380AGAGTATAACCTTCCATTTT 50 10333 10352 1306 560433 1365 1384TTATAGAGTATAACCTTCCA 63 10337 10356 1307 560434 1369 1388GATTTTATAGAGTATAACCT 31 10341 10360 1308 560435 1370 1389TGATTTTATAGAGTATAACC 6 10342 10361 1309 560436 1371 1390TTGATTTTATAGAGTATAAC 14 10343 10362 1310 560437 1372 1391GTTGATTTTATAGAGTATAA 2 10344 10363 1311 560438 1376 1395TTTGGTTGATTTTATAGAGT 20 10348 10367 1312 560439 1386 1405GGATCAACATTTTGGTTGAT 42 10358 10377 1313 560440 1387 1406TGGATCAACATTTTGGTTGA 10 10359 10378 1314 560441 1388 1407ATGGATCAACATTTTGGTTG 34 10360 10379 1315 560442 1398 1417AATCTGTTGGATGGATCAAC 52 10370 10389 1316 560443 1399 1418GAATCTGTTGGATGGATCAA 47 10371 10390 1317 560444 1403 1422TTCTGAATCTGTTGGATGGA 30 10375 10394 1318 560445 1404 1423TTTCTGAATCTGTTGGATGG 34 10376 10395 1319 560446 1405 1424CTTTCTGAATCTGTTGGATG 50 10377 10396 1320 560447 1409 1428AAAGCTTTCTGAATCTGTTG 29 10381 10400 1321 560448 1425 1444TTGCCTCAGTTCATTCAAAG 38 10397 10416 1322 560449 1429 1448AAATTTGCCTCAGTTCATTC 27 10401 10420 1323 560450 1434 1453CTTTTAAATTTGCCTCAGTT 34 10406 10425 1324 560451 1440 1459TATTGCCTTTTAAATTTGCC 21 10412 10431 1325 560452 1441 1460TTATTGCCTTTTAAATTTGC 23 10413 10432 1326 560453 1446 1465TTAAATTATTGCCTTTTAAA 1 10418 10437 1327 560454 1447 1466TTTAAATTATTGCCTTTTAA 1 10419 10438 1328 560455 1448 1467GTTTAAATTATTGCCTTTTA 48 10420 10439 1329 560456 1449 1468TGTTTAAATTATTGCCTTTT 25 10421 10440 1330 560457 1450 1469ATGTTTAAATTATTGCCTTT 0 10422 10441 1331 560458 1704 1723TTTAATAAGTTCACCTATTG 26 10676 10695 1332 560459 1705 1724ATTTAATAAGTTCACCTATT 26 10677 10696 1333 560460 1706 1725TATTTAATAAGTTCACCTAT 16 10678 10697 1334 560461 1707 1726TTATTTAATAAGTTCACCTA 4 10679 10698 1335 560462 1708 1727GTTATTTAATAAGTTCACCT 36 10680 10699 1336 560463 1709 1728AGTTATTTAATAAGTTCACC 0 10681 10700 1337 560464 1712 1731AAAAGTTATTTAATAAGTTC 12 10684 10703 1338 560465 1719 1738TATTTAGAAAAGTTATTTAA 0 10691 10710 1339 560466 1738 1757TAAAAGTCTCTAAATTTTTT 0 10710 10729 1340 560467 1739 1758ATAAAAGTCTCTAAATTTTT 0 10711 10730 1341 560468 1740 1759AATAAAAGTCTCTAAATTTT 25 10712 10731 1342 560469 1760 1779GCTCATATGATGCCTTTTAA 77 10732 10751 38 560470 1761 1780AGCTCATATGATGCCTTTTA 73 10733 10752 39 560471 1762 1781TAGCTCATATGATGCCTTTT 67 10734 10753 40 560472 1763 1782TTAGCTCATATGATGCCTTT 42 10735 10754 1343 560473 1764 1783ATTAGCTCATATGATGCCTT 61 10736 10755 1344 560474 1765 1784TATTAGCTCATATGATGCCT 55 10737 10756 41 560475 1766 1785ATATTAGCTCATATGATGCC 42 10738 10757 1345 560476 1767 1786GATATTAGCTCATATGATGC 36 10739 10758 1346 560477 1768 1787TGATATTAGCTCATATGATG 21 10740 10759 1347 560478 1769 1788GTGATATTAGCTCATATGAT 40 10741 10760 1348 560479 1776 1795GAAAGTTGTGATATTAGCTC 43 10748 10767 1349 560480 1777 1796GGAAAGTTGTGATATTAGCT 19 10749 10768 1350 560481 1778 1797GGGAAAGTTGTGATATTAGC 17 10750 10769 1351 560482 1779 1798TGGGAAAGTTGTGATATTAG 29 10751 10770 1352 560483 1780 1799CTGGGAAAGTTGTGATATTA 35 10752 10771 1353 560484 1781 1800ACTGGGAAAGTTGTGATATT 25 10753 10772 1354 560485 1782 1801AACTGGGAAAGTTGTGATAT 12 10754 10773 1355 560486 1783 1802AAACTGGGAAAGTTGTGATA 21 10755 10774 1356 560487 1784 1803TAAACTGGGAAAGTTGTGAT 22 10756 10775 1357 560488 1785 1804TTAAACTGGGAAAGTTGTGA 12 10757 10776 1358 560489 1786 1805TTTAAACTGGGAAAGTTGTG 22 10758 10777 1359 560490 1787 1806TTTTAAACTGGGAAAGTTGT 23 10759 10778 1360 560491 1790 1809GTTTTTTAAACTGGGAAAGT 1 10762 10781 1361 560492 1791 1810AGTTTTTTAAACTGGGAAAG 0 10763 10782 1362 560493 1792 1811TAGTTTTTTAAACTGGGAAA 0 10764 10783 1363 560494 1796 1815GTACTAGTTTTTTAAACTGG 23 10768 10787 1364 560495 1799 1818AGAGTACTAGTTTTTTAAAC 0 10771 10790 1365 560496 1801 1820CAAGAGTACTAGTTTTTTAA 0 10773 10792 1366 560497 1806 1825TTTAACAAGAGTACTAGTTT 21 10778 10797 1367 560498 1807 1826TTTTAACAAGAGTACTAGTT 19 10779 10798 1368 560499 1808 1827GTTTTAACAAGAGTACTAGT 37 10780 10799 1369 560500 1809 1828AGTTTTAACAAGAGTACTAG 20 10781 10800 1370 560501 1810 1829GAGTTTTAACAAGAGTACTA 21 10782 10801 1371 560502 1811 1830AGAGTTTTAACAAGAGTACT 0 10783 10802 1372 560503 1814 1833TTTAGAGTTTTAACAAGAGT 0 10786 10805 1373 560504 1815 1834GTTTAGAGTTTTAACAAGAG 18 10787 10806 1374 560505 1817 1836AAGTTTAGAGTTTTAACAAG 9 10789 10808 1375 560506 1818 1837CAAGTTTAGAGTTTTAACAA 1 10790 10809 1376 560507 1822 1841TAGTCAAGTTTAGAGTTTTA 21 10794 10813 1377 560508 1823 1842TTAGTCAAGTTTAGAGTTTT 10 10795 10814 1378 560509 1824 1843TTTAGTCAAGTTTAGAGTTT 20 10796 10815 1379 560510 1828 1847TGTATTTAGTCAAGTTTAGA 8 10800 10819 1380 560511 1829 1848CTGTATTTAGTCAAGTTTAG 37 10801 10820 1381 560512 1830 1849TCTGTATTTAGTCAAGTTTA 46 10802 10821 1382 560513 1834 1853GTCCTCTGTATTTAGTCAAG 38 10806 10825 1383 560514 1835 1854AGTCCTCTGTATTTAGTCAA 29 10807 10826 1384 560515 1836 1855CAGTCCTCTGTATTTAGTCA 47 10808 10827 1385 560516 1837 1856CCAGTCCTCTGTATTTAGTC 31 10809 10828 1386 560517 1838 1857ACCAGTCCTCTGTATTTAGT 31 10810 10829 1387 560518 1839 1858TACCAGTCCTCTGTATTTAG 35 10811 10830 1388 560519 1840 1859TTACCAGTCCTCTGTATTTA 30 10812 10831 1389 560520 1841 1860ATTACCAGTCCTCTGTATTT 37 10813 10832 1390 560521 1842 1861AATTACCAGTCCTCTGTATT 12 10814 10833 1391 560522 1843 1862CAATTACCAGTCCTCTGTAT 38 10815 10834 1392 560523 1844 1863ACAATTACCAGTCCTCTGTA 35 10816 10835 1393 560524 1845 1864TACAATTACCAGTCCTCTGT 51 10817 10836 1394 560525 1846 1865GTACAATTACCAGTCCTCTG 52 10818 10837 1395 560526 1847 1866TGTACAATTACCAGTCCTCT 38 10819 10838 1396 560527 1848 1867CTGTACAATTACCAGTCCTC 19 10820 10839 1397 560528 1849 1868ACTGTACAATTACCAGTCCT 13 10821 10840 1398 560529 1850 1869AACTGTACAATTACCAGTCC 27 10822 10841 1399 560530 1851 1870GAACTGTACAATTACCAGTC 20 10823 10842 1400 560531 1852 1871AGAACTGTACAATTACCAGT 24 10824 10843 1401 560532 1854 1873TAAGAACTGTACAATTACCA 22 10826 10845 1402 560533 1855 1874TTAAGAACTGTACAATTACC 20 10827 10846 1403 560534 1856 1875TTTAAGAACTGTACAATTAC 1 10828 10847 1404

TABLE 133 Inhibition of ANGPTL3 mRNA by 5-10-5 MOE gapmers targeting SEQID NO: 1 and 2 SEQ SEQ SEQ ID SEQ ID ID NO: NO: ID NO: NO: 2 SEQ 1 Start1 Stop % 2 Start Stop ID ISIS NO Site Site Sequence inhibition Site SiteNO 544355 N/A N/A TTTCAGCATGTATCTCTTAA 69 7065 7084 21 544376 N/A N/AGGAGTGGTTCTTTTCACAGC 64 8298 8317 24 544380 N/A N/A TGGTCCTTTTAACTTCCAAT50 8365 8384 25 560612 N/A N/A ACTTGAAATTATAATAGGAA 0 3798 3817 1405560613 N/A N/A AAAAAACTAACTTGAAATTA 0 3807 3826 1406 560614 N/A N/AGAAACAAAAAACTAACTTGA 21 3812 3831 1407 560615 N/A N/AGTGTTTTCATATATAACATT 19 3835 3854 1408 560616 N/A N/AAATTTTCAGTGTTTTCATAT 0 3843 3862 1409 560617 N/A N/AAAAATGCAAATTTTCAGTGT 0 3851 3870 1410 560618 N/A N/AGTAATTTTCATATAAAATGC 0 3864 3883 1411 560619 N/A N/AGATTTGTAATTTTCATATAA 0 3869 3888 1412 560620 N/A N/ATAACCGATTTGTAATTTTCA 16 3874 3893 1413 560621 N/A N/ATAATTTAACCGATTTGTAAT 5 3879 3898 1414 560622 N/A N/ATTGTATAATTTAACCGATTT 13 3884 3903 1415 560623 N/A N/ACTAGATTGTATAATTTAACC 8 3889 3908 1416 560624 N/A N/AGTGTTCTAGATTGTATAATT 24 3894 3913 1417 560625 N/A N/AAATGACATAGTGTTCTAGAT 0 3903 3922 1418 560626 N/A N/AAGTGTAATGACATAGTGTTC 10 3908 3927 1419 560627 N/A N/ATTACAATAGTGTAATGACAT 0 3915 3934 1420 560628 N/A N/ATTCAGTAATTTACAATAGTG 12 3924 3943 1421 560629 N/A N/ATTACCTTCAGTAATTTACAA 9 3929 3948 1422 560630 N/A N/ATTAACTTTTTACTTACCTTC 7 3941 3960 1423 560631 N/A N/AGAATAGTTTTAAATTTTTTT 0 3960 3979 1424 560632 N/A N/AACACTGGAGAATAGTTTTAA 10 3968 3987 1425 560633 N/A N/ATTTAAACACTGGAGAATAGT 0 3973 3992 1426 560634 N/A N/ATCTGTTTTAAACACTGGAGA 25 3978 3997 1427 560635 N/A N/AGTATTATTTAATCTGTTTTA 0 3989 4008 1428 560636 N/A N/ATTACTGTATTATTTAATCTG 5 3994 4013 1429 560637 N/A N/ATAAATCTTTTCCATTTACTG 18 4008 4027 1430 560638 N/A N/AATGAATAAATCTTTTCCATT 12 4013 4032 1431 560639 N/A N/AGCATATTTTCATATGAATAA 9 4025 4044 1432 560640 N/A N/AGCCCAGCATATTTTCATATG 20 4030 4049 1433 560641 N/A N/AAAAAGAAAAAGCCCAGCATA 20 4040 4059 1434 560642 N/A N/ACTGAACTTCAATTAAAAGAA 5 4053 4072 1435 560643 N/A N/AGATTTTCTGAACTTCAATTA 9 4059 4078 1436 560644 N/A N/ATCTAAAATTTGATTTTCTGA 0 4069 4088 1437 560645 N/A N/AACTATCTCTAAAATTTGATT 8 4075 4094 1438 560646 N/A N/ATTAAATTGTACTATCTCTAA 5 4084 4103 1439 560647 N/A N/AACATTTTATTTAAATTGTAC 17 4093 4112 1440 560648 N/A N/AGTCCTTAACATTTTATTTAA 0 4100 4119 1441 560649 N/A N/ACATATTTTTGTCCTTAACAT 0 4109 4128 1442 560650 N/A N/ATAGCACATATTTTTGTCCTT 25 4114 4133 1443 560651 N/A N/ATCAAATAGCACATATTTTTG 0 4119 4138 1444 560652 N/A N/ACTTCTTTCAAATAGCACATA 41 4125 4144 1445 560653 N/A N/ACTTGTATGCTTCTTTCAAAT 19 4133 4152 1446 560654 N/A N/AATTCCTTCCCCTTGTATGCT 12 4143 4162 1447 560655 N/A N/ATTGGCAATTCCTTCCCCTTG 36 4149 4168 1448 560656 N/A N/AGAATATTGGCAATTCCTTCC 38 4154 4173 1449 560657 N/A N/ATGAAAAATGAATATTGGCAA 0 4162 4181 1450 560658 N/A N/ATAATGGATTTGAAAAATGAA 0 4171 4190 1451 560659 N/A N/AACTAATAATGGATTTGAAAA 1 4176 4195 1452 560660 N/A N/ACATAATCTAAATTTTTAAAC 6 4194 4213 1453 560661 N/A N/ACACTATCATAATCTAAATTT 4 4200 4219 1454 560662 N/A N/AAATTTCCTGTAACACTATCA 2 4212 4231 1455 560663 N/A N/ACTATTAATTTCCTGTAACAC 9 4217 4236 1456 560664 N/A N/ACTTTTCTATTAATTTCCTGT 5 4222 4241 1457 560665 N/A N/ACTCTTTCTTTTCTATTAATT 0 4228 4247 1458 560666 N/A N/AAGTTGCTTTCCTCTTTCTTT 0 4238 4257 1459 560667 N/A N/ATTATAAGTTGCTTTCCTCTT 10 4243 4262 1460 560668 N/A N/AGTTGGTTATAAGTTGCTTTC 6 4248 4267 1461 560669 N/A N/AAGTAGGTTGGTTATAAGTTG 4 4253 4272 1462 560670 N/A N/ATAGAGAGTAGGTTGGTTATA 0 4258 4277 1463 560671 N/A N/AGGATATAGAGAGTAGGTTGG 0 4263 4282 1464 560672 N/A N/AAGTCTGGATATAGAGAGTAG 0 4268 4287 1465 560673 N/A N/ATACAAAAGTCTGGATATAGA 7 4274 4293 1466 560674 N/A N/AGTTTTTCTACAAAAGTCTGG 12 4281 4300 1467 560675 N/A N/ATTACCTGATTTTCTATTTCT 15 4380 4399 1468 560676 N/A N/AATACTGACTTACCTGATTTT 15 4388 4407 1469 560677 N/A N/ATTAAAATACTGACTTACCTG 2 4393 4412 1470 560678 N/A N/ATACCATTAAAATACTGACTT 0 4398 4417 1471 560679 N/A N/AGGACATACCATTAAAATACT 7 4403 4422 1472 560680 N/A N/AAAAGATGGGACATACCATTA 0 4410 4429 1473 560681 N/A N/AAGACCTGTGTGAAAGATGGG 19 4421 4440 1474 560682 N/A N/ATTTACAGACCTGTGTGAAAG 22 4426 4445 1475 560683 N/A N/AGTGTTTTTACAGACCTGTGT 47 4431 4450 1476 560684 N/A N/AATTCAGTGTTTTTACAGACC 44 4436 4455 1477 560685 N/A N/ATTAGGATTCAGTGTTTTTAC 46 4441 4460 1478 560686 N/A N/AATAATTTTAGGATTCAGTGT 15 4447 4466 1479 560687 N/A N/AGCTTGTAAATAATTTTAGGA 0 4455 4474 1480 560688 N/A N/AGTTAAAGCTTGTAAATAATT 0 4461 4480 1481 560689 N/A N/ATGTTTTATATCTCTTGAAAA 0 5571 5590 1482 560690 N/A N/ATTGGTAATAATATTTGTTTT 9 5585 5604 1483 560691 N/A N/AGGAAATTGGTAATAATATTT 0 5590 5609 1484 560692 N/A N/ATTAGTGGAAATTGGTAATAA 22 5595 5614 1485 560693 N/A N/ATTTGTTTAGTGGAAATTGGT 8 5600 5619 1486 560694 N/A N/ATTATGTTTGTTTAGTGGAAA 0 5605 5624 1487 560695 N/A N/ATAACATTATGTTTGTTTAGT 12 5610 5629 1488 560696 N/A N/AACTACTAACATTATGTTTGT 4 5615 5634 1489 560697 N/A N/AGCAGCACTACTAACATTATG 38 5620 5639 1490 560698 N/A N/ATTTTAGCAGCACTACTAACA 15 5625 5644 1491 560699 N/A N/AAAACCTTTTAGCAGCACTAC 52 5630 5649 1492 560700 N/A N/AGATAAAAAACCTTTTAGCAG 0 5636 5655 1493 560701 N/A N/ATAGTTGATAAAAAACCTTTT 0 5641 5660 1494 560702 N/A N/ACAAAAGTAGTTGATAAAAAA 0 5647 5666 1495 560703 N/A N/AATGGAAACCAAAAGTAGTTG 13 5655 5674 1496 560704 N/A N/AAAAGTATGGAAACCAAAAGT 20 5660 5679 1497 560705 N/A N/AGAAGGAAAGTATGGAAACCA 45 5665 5684 1498 560706 N/A N/ACATAAGAAGGAAAGTATGGA 10 5670 5689 1499 560707 N/A N/ATAACATCATAAGAAGGAAAG 0 5676 5695 1500 560708 N/A N/AGAATAATAACATCATAAGAA 0 5682 5701 1501 560709 N/A N/AGAATTTAGAATAATAACATC 1 5689 5708 1502 560710 N/A N/ATATAATTGAAAAGAATTTAG 8 5701 5720 1503 560711 N/A N/ATAGTAAAAGATATAATTGAA 0 5711 5730 1504 560712 N/A N/AAATCATAGTAAAAGATATAA 10 5716 5735 1505 560713 N/A N/ACAGGTTCATTTAATCATAGT 43 5727 5746 1506 560714 N/A N/ACTATAGTAACATTTTGCTTT 24 5753 5772 1507 560715 N/A N/AGTATATTACTATAGTAACAT 18 5761 5780 1508 560716 N/A N/AACAATGTATATTACTATAGT 0 5766 5785 1509 560717 N/A N/ATAGACACAATGTATATTACT 46 5771 5790 1510 560718 N/A N/ATATTTTTAGACACAATGTAT 29 5777 5796 1511 560719 N/A N/AACACATTTTTATTTTTAGAC 15 5786 5805 1512 560720 N/A N/ATTGGTTTCTTCACACATTTT 62 5797 5816 1513 560721 N/A N/ATTCATTGTTTTGGTTTCTTC 55 5806 5825 1514 560722 N/A N/ACAGAAATTCATTGTTTTGGT 55 5812 5831 1515 560723 N/A N/ATCCAACTCAGAAATTCATTG 65 5819 5838 48 560724 N/A N/A CTTCTTCCAACTCAGAAATT41 5824 5843 1516 560725 N/A N/A TGATCTAACTCTTCTTCCAA 24 5834 5853 1517560726 N/A N/A TTAAATGATCTAACTCTTCT 23 5839 5858 1518 560727 N/A N/ATGAGAAAGTTAAATGATCTA 0 5847 5866 1519 560728 N/A N/ATACTTAAATTTTTAGAGTTT 10 5886 5905 1520 560729 N/A N/AAAAGTTACTTAAATTTTTAG 3 5891 5910 1521 560730 N/A N/AATCTTAAAGTTACTTAAATT 0 5896 5915 1522 560731 N/A N/AATGTGATCTTAAAGTTACTT 24 5901 5920 1523 560732 N/A N/ATAACTATGTGATCTTAAAGT 0 5906 5925 1524 560733 N/A N/ATTACTCTTTTCTACTAAGTA 39 5924 5943 1525 560734 N/A N/AGGGTATTACTCTTTTCTACT 48 5929 5948 1526 560735 N/A N/ATTGCTGGGTATTACTCTTTT 75 5934 5953 49 560736 N/A N/A TTTGCTTGCTGGGTATTACT65 5939 5958 50 560737 N/A N/A TAAAGTTTGCTTGCTGGGTA 49 5944 5963 1527560738 N/A N/A TATTGTAAAGTTTGCTTGCT 15 5949 5968 1528 560739 N/A N/ATAAAAGGATCTATTGTAAAG 0 5959 5978 1529 560740 N/A N/ATTATTTAAAAGGATCTATTG 9 5964 5983 1530 560741 N/A N/AGGACCTTATTTAAAAGGATC 17 5969 5988 1531 560742 N/A N/AGATATTTCCTAGGACCTTAT 27 5980 5999 1532 560743 N/A N/ATGAATGATATTTCCTAGGAC 0 5985 6004 1533 560744 N/A N/ATGGCATGAATGATATTTCCT 74 5990 6009 51 560745 N/A N/A GATGCTGGCATGAATGATAT40 5995 6014 1534 560746 N/A N/A TTTTTTGATGCTGGCATGAA 38 6001 6020 1535560747 N/A N/A GTTAGTTTTTTGATGCTGGC 35 6006 6025 1536 560748 N/A N/ATTAGTGTTAGTTTTTTGATG 0 6011 6030 1537 560749 N/A N/AGCATTATTAGTGTTAGTTTT 50 6017 6036 1538 560750 N/A N/AATCTTGCATTATTAGTGTTA 49 6022 6041 1539 560751 N/A N/AATAATATCTTGCATTATTAG 17 6027 6046 1540 560752 N/A N/ACAGTAAGAAAAGCAGAATAT 15 6047 6066 1541 560753 N/A N/ATCATTGACAGTAAGAAAAGC 47 6054 6073 1542 560754 N/A N/AGATAGTTTTTCTCATTGACA 40 6065 6084 1543 560755 N/A N/AGTTTGCAATTTATTGAATGA 12 6083 6102 1544 560756 N/A N/AGTGTTGGGTTTGCAATTTAT 55 6090 6109 1545 560757 N/A N/ATTAAGTGTGTTGGGTTTGCA 50 6096 6115 1546 560758 N/A N/ATTTTATTTAAGTGTGTTGGG 5 6102 6121 1547 560759 N/A N/ATTTAGCAGTAACATTTTATT 19 6121 6140 1548 560760 N/A N/AGTTAGTTTAGCAGTAACATT 30 6126 6145 1549 560761 N/A N/ATCTATATATTCAGTAGTTTA 17 6148 6167 1550 560762 N/A N/ATTACTTTCTATATATTCAGT 14 6154 6173 1551 560763 N/A N/AGTTTGCTTACTTTCTATATA 20 6160 6179 1552 560764 N/A N/AAGTTTGTTTGCTTACTTTCT 36 6165 6184 1553 560765 N/A N/ATGGCAAGTTTGTTTGCTTAC 43 6170 6189 1554 560766 N/A N/ATTACTGTTACTGTATTTCCC 39 10155 10174 1555 560767 N/A N/AATGTAGTTACTGTTACTGTA 18 10161 10180 1556 560768 N/A N/AATTTAATGGGTACAGACTCG 47 10182 10201 61 560769 N/A N/AATGCAATTTAATGGGTACAG 32 10187 10206 1557 560770 N/A N/ATAGATATGCAATTTAATGGG 4 10192 10211 1558 560771 N/A N/AAGGAGATAGATATGCAATTT 5 10198 10217 1559 560772 N/A N/ACCTAAAGGAGATAGATATGC 36 10203 10222 1560 560773 N/A N/AAGCCTCCTAAAGGAGATAGA 0 10208 10227 1561 560774 N/A N/ACACCACCAGCCTCCTAAAGG 35 10215 10234 1562 560775 N/A N/AATCTAAGAAAATTAATAAAC 17 7003 7022 1563 560776 N/A N/AATGATCACATCTAAGAAAAT 8 7011 7030 1564 560777 N/A N/AATACCATGATCACATCTAAG 49 7016 7035 62 560778 N/A N/A GCAATACCATGATCACATCT59 7019 7038 52 560779 N/A N/A AACTGCAATACCATGATCAC 35 7023 7042 1565560780 N/A N/A TAAAACTGCAATACCATGAT 43 7026 7045 1566 560781 N/A N/ACTTTAAAACTGCAATACCAT 13 7029 7048 1567 560782 N/A N/ATCTCCTTTAAAACTGCAATA 18 7033 7052 1568 560783 N/A N/ATGTTCTCCTTTAAAACTGCA 13 7036 7055 1569 560784 N/A N/AGATTGTTCTCCTTTAAAACT 23 7039 7058 1570 560785 N/A N/AAGGAGATTGTTCTCCTTTAA 14 7043 7062 1571 560786 N/A N/AAACAGGAGATTGTTCTCCTT 0 7046 7065 1572 560787 N/A N/ATTAAACAGGAGATTGTTCTC 7 7049 7068 1573 560788 N/A N/ACTCTTAAACAGGAGATTGTT 10 7052 7071 1574 560789 N/A N/AACTCCGTAAATATTTCAGCA 55 7077 7096 53 560790 N/A N/A CTTTAACTCCGTAAATATTT22 7082 7101 1575 560791 N/A N/A GACCTTTAACTCCGTAAATA 54 7085 7104 63560792 N/A N/A AGTGACCTTTAACTCCGTAA 35 7088 7107 1576 560793 N/A N/AGGAGTCCAGTGACCTTTAAC 15 7095 7114 1577 560794 N/A N/ATCTGGAGTCCAGTGACCTTT 46 7098 7117 64 560795 N/A N/A ACCAGTCTGGAGTCCAGTGA8 7103 7122 1578 560796 N/A N/A TCATCTTACCAAACTATTTT 22 7169 7188 1579560797 N/A N/A GAATCATCTTACCAAACTAT 39 7172 7191 1580 560798 N/A N/ATAAGAATCATCTTACCAAAC 35 7175 7194 1581 560799 N/A N/AATGTAAGAATCATCTTACCA 52 7178 7197 65 560800 N/A N/A AAGAATGTAAGAATCATCTT22 7182 7201 1582 560801 N/A N/A GTTATTTAAGAATGTAAGAA 0 7189 7208 1583560802 N/A N/A CGTGTTATTTAAGAATGTAA 3 7192 7211 1584 560803 N/A N/AAGCATTTTTCTTAGATGGCG 48 7210 7229 66 560804 N/A N/A TAAAGCATTTTTCTTAGATG0 7213 7232 1585 560805 N/A N/A TGTTAAAGCATTTTTCTTAG 0 7216 7235 1586560806 N/A N/A TTTATGTTAAAGCATTTTTC 20 7220 7239 1587 560807 N/A N/AATGTTTATGTTAAAGCATTT 8 7223 7242 1588 560808 N/A N/AGCATTTTTTCAGTAATGTTT 40 7237 7256 1589 560809 N/A N/ATGTAGCATTTTTTCAGTAAT 24 7241 7260 1590 560810 N/A N/ACAAATGTAGCATTTTTTCAG 0 7245 7264 1591 560811 N/A N/ATGGCAAATGTAGCATTTTTT 60 7248 7267 54 560812 N/A N/A AAGTTGTGGCAAATGTAGCA26 7254 7273 1592 560813 N/A N/A ATGAAGTTGTGGCAAATGTA 11 7257 7276 1593560814 N/A N/A TTTATGAAGTTGTGGCAAAT 36 7260 7279 1594 560815 N/A N/ACATTTTATGAAGTTGTGGCA 45 7263 7282 67 560816 N/A N/A TGACATTTTATGAAGTTGTG16 7266 7285 1595 560817 N/A N/A CACTTGACATTTTATGAAGT 47 7270 7289 68560818 N/A N/A CTTGAGATTTCACTTGACAT 18 7280 7299 1596 560819 N/A N/ATTTGGAGCTTGAGATTTCAC 0 7287 7306 1597 560820 N/A N/AATCTTTGGAGCTTGAGATTT 0 7290 7309 1598 560821 N/A N/AAATATCTTTGGAGCTTGAGA 6 7293 7312 1599 560822 N/A N/AAATAATATCTTTGGAGCTTG 24 7296 7315 1600 560823 N/A N/AAGGAATAATATCTTTGGAGC 1 7299 7318 1601 560824 N/A N/AAATAGGAATAATATCTTTGG 0 7302 7321 1602 560825 N/A N/AAGTAATAGGAATAATATCTT 0 7305 7324 1603 560826 N/A N/ATTACATCAGATTTAGTAATA 0 7318 7337 1604 560827 N/A N/AAAATGTTATTACATCAGATT 0 7326 7345 1605 560828 N/A N/AATAAAATGTTATTACATCAG 12 7329 7348 1606 560829 N/A N/ACCTAGAATCAATAAAATGTT 13 7339 7358 1607 560830 N/A N/AAGGAATGCCTAGAATCAATA 9 7346 7365 1608 560831 N/A N/AATTCAGCAGGAATGCCTAGA 26 7353 7372 1609 560832 N/A N/ATACATTCAGCAGGAATGCCT 23 7356 7375 1610 560833 N/A N/ATTACCTGATATAACATCACA 30 7456 7475 1611 560834 N/A N/AGTTTTACCTGATATAACATC 6 7459 7478 1612 560835 N/A N/ACAGGTTTTACCTGATATAAC 4 7462 7481 1613 560836 N/A N/ATTAGACAGGTTTTACCTGAT 6 7467 7486 1614 560837 N/A N/AATTCTCCTTAGACAGGTTTT 6 7474 7493 1615 560838 N/A N/AACTGTCTATTCTCCTTAGAC 0 7481 7500 1616 560839 N/A N/AACTACTGTCTATTCTCCTTA 17 7484 7503 1617 560840 N/A N/AACTAACTACTGTCTATTCTC 0 7488 7507 1618 560841 N/A N/ATGAACTAACTACTGTCTATT 0 7491 7510 1619 560842 N/A N/AAGTTGAACTAACTACTGTCT 0 7494 7513 1620 560844 N/A N/AATTAATTGATATGTAAAACG 0 8347 8366 1621 560845 N/A N/ACCAATTAATTGATATGTAAA 15 8350 8369 1622 560846 N/A N/ATCCTTTTAACTTCCAATTAA 29 8362 8381 1623 560847 N/A N/ATCCTGGTCCTTTTAACTTCC 58 8368 8387 69 560848 N/A N/A GTTTCCTGGTCCTTTTAACT0 8371 8390 1624 560849 N/A N/A TCTGAGTTTCCTGGTCCTTT 36 8376 8395 1625560850 N/A N/A ATGTCTGAGTTTCCTGGTCC 31 8379 8398 1626 560851 N/A N/ATGTATGTCTGAGTTTCCTGG 0 8382 8401 1627 560852 N/A N/AATGTATACTGTATGTCTGAG 19 8390 8409 1628 560853 N/A N/AAAAATGTATACTGTATGTCT 12 8393 8412 1629 560854 N/A N/ATTTTAAAATGTATACTGTAT 0 8397 8416 1630 560855 N/A N/ACATACATTCTATATATTATA 29 8432 8451 1631 560856 N/A N/AAAGCCATACATTCTATATAT 38 8436 8455 55 560857 N/A N/A ATTATAAGCCATACATTCTA6 8441 8460 1632 560858 N/A N/A TTCATTATAAGCCATACATT 0 8444 8463 1633560859 N/A N/A TAATTCATTATAAGCCATAC 19 8447 8466 1634 560860 N/A N/ATGAGTTAACTAATTCATTAT 0 8456 8475 1635 560861 N/A N/ATTTGCATTGAGTTAACTAAT 26 8463 8482 1636 560862 N/A N/ATAATTTGCATTGAGTTAACT 0 8466 8485 1637 560863 N/A N/AGAATAATTTGCATTGAGTTA 0 8469 8488 1638 560864 N/A N/AATAGAATAATTTGCATTGAG 0 8472 8491 1639 560865 N/A N/AAAAATAGAATAATTTGCATT 0 8475 8494 1640 560866 N/A N/ATTGTAATCAAAATAGAATAA 0 8483 8502 1641 560867 N/A N/ATATTTGTAATCAAAATAGAA 16 8486 8505 1642 560868 N/A N/ATACTATTTGTAATCAAAATA 0 8489 8508 1643 560869 N/A N/ATTTTACTATTTGTAATCAAA 0 8492 8511 1644 560870 N/A N/AGCTTATTTTACTATTTGTAA 0 8497 8516 1645 560871 N/A N/ACTTGCTTATTTTACTATTTG 0 8500 8519 1646 560872 N/A N/ATTATCTTGCTTATTTTACTA 1 8504 8523 1647 560873 N/A N/AGTTATTTTATCTTGCTTATT 0 8510 8529 1648 560874 N/A N/AAAACATCTGTTATTTTATCT 0 8518 8537 1649 560875 N/A N/AGGATTTTAAACATCTGTTAT 0 8525 8544 1650 560876 N/A N/ACTTTTTGGATTTTAAACATC 24 8531 8550 1651 560877 N/A N/AGTGCTTTTTGGATTTTAAAC 6 8534 8553 1652 560878 N/A N/ATTTTGTATGTGCTTTTTGGA 24 8542 8561 1653 560879 N/A N/AGACATCATTCATGGATTTTT 50 8558 8577 70 560880 N/A N/A AGTACTTAGACATCATTCAT43 8566 8585 71 560881 N/A N/A TAAGTGAGTACTTAGACATC 17 8572 8591 1654560882 N/A N/A TACTTTATAAGTGAGTACTT 0 8579 8598 1655 560883 N/A N/ATTCTACTTTATAAGTGAGTA 32 8582 8601 1656 560884 N/A N/AAATGTCTTCTACTTTATAAG 0 8588 8607 1657 560885 N/A N/AAATAATGAATGTCTTCTACT 9 8595 8614 1658 560886 N/A N/ATATAATAATGAATGTCTTCT 0 8598 8617 1659 560887 N/A N/ATGATATAATAATGAATGTCT 29 8601 8620 1660 560888 N/A N/AAAAATTTGATATAATAATGA 0 8607 8626 1661 560889 N/A N/ACATTTAAAAATTTGATATAA 0 8613 8632 1662 560890 N/A N/AGTACTGAGCATTTAAAAATT 8 8621 8640 1663 560891 N/A N/AGGTCAAATAGTACTGAGCAT 40 8630 8649 72 560892 N/A N/A AATGGTCAAATAGTACTGAG23 8633 8652 1664 560893 N/A N/A TTAAATGGTCAAATAGTACT 17 8636 8655 1665560894 N/A N/A AGTTTGAATACAAAATTTTT 0 8654 8673 1666 560895 N/A N/AGGTAGTTTGAATACAAAATT 38 8657 8676 73 560896 N/A N/A ACTGGTAGTTTGAATACAAA0 8660 8679 1667 560897 N/A N/A TTCACTGGTAGTTTGAATAC 0 8663 8682 1668560898 N/A N/A GCTTTCACTGGTAGTTTGAA 25 8666 8685 1669 560899 N/A N/AAGGGCTTTCACTGGTAGTTT 30 8669 8688 1670 560900 N/A N/AGGTAGGGCTTTCACTGGTAG 9 8672 8691 1671 560901 N/A N/ACTAGGTAGGGCTTTCACTGG 37 8675 8694 1672 560902 N/A N/ACTTCTAGGTAGGGCTTTCAC 32 8678 8697 1673 560903 N/A N/ATACCTTCTAGGTAGGGCTTT 26 8681 8700 1674 560904 N/A N/AGTATACCTTCTAGGTAGGGC 0 8684 8703 1675 560905 N/A N/ATGAGTATACCTTCTAGGTAG 15 8687 8706 1676 560906 N/A N/ACACTGAGTATACCTTCTAGG 36 8690 8709 1677 560907 N/A N/ATATCACTGAGTATACCTTCT 0 8693 8712 1678 560908 N/A N/AACTTATCACTGAGTATACCT 28 8696 8715 1679 560909 N/A N/AACAAAACTTATCACTGAGTA 32 8701 8720 1680 560910 N/A N/AGCTACAAAACTTATCACTGA 15 8704 8723 1681 560911 N/A N/AGGAGCTACAAAACTTATCAC 21 8707 8726 1682 560912 N/A N/AGATTTGGAGCTACAAAACTT 0 8712 8731 1683 560913 N/A N/AGAAGATTTGGAGCTACAAAA 0 8715 8734 1684 560914 N/A N/ACTATTAGAAGATTTGGAGCT 0 8721 8740 1685 560915 N/A N/ACACTCACTATTAGAAGATTT 33 8727 8746 1686 560916 N/A N/ATGTCAGCCTTTTATTTTGGG 0 8751 8770 1687 560917 N/A N/AACCTGTCAGCCTTTTATTTT 11 8754 8773 1688 560918 N/A N/ATCGACTTACCTGTCAGCCTT 0 8761 8780 1689 560919 N/A N/ATTCTCGACTTACCTGTCAGC 0 8764 8783 1690 560920 N/A N/AGTATTCTCGACTTACCTGTC 0 8767 8786 1691 560921 N/A N/ATAACATCCATATACAGTCAA 25 9177 9196 1692 560922 N/A N/ATATTAACATCCATATACAGT 20 9180 9199 1693 560923 N/A N/AATTTATTAACATCCATATAC 20 9183 9202 1694 560924 N/A N/AGCTATTTATTAACATCCATA 47 9186 9205 1695 560925 N/A N/ATCAGCTATTTATTAACATCC 58 9189 9208 56 560926 N/A N/A CTGTCAGCTATTTATTAACA30 9192 9211 1696 560927 N/A N/A TTACTGTCAGCTATTTATTA 22 9195 9214 1697560928 N/A N/A ACTTTACTGTCAGCTATTTA 27 9198 9217 1698 560929 N/A N/ATAAACTTTACTGTCAGCTAT 41 9201 9220 1699 560930 N/A N/AGGATAAACTTTACTGTCAGC 45 9204 9223 1700 560931 N/A N/ATATGGATAAACTTTACTGTC 15 9207 9226 1701 560932 N/A N/ATTATATGGATAAACTTTACT 0 9210 9229 1702 560933 N/A N/ATTGCAAGTCTTTATATGGAT 47 9220 9239 1703 560934 N/A N/ATATTTGCAAGTCTTTATATG 26 9223 9242 1704 560935 N/A N/AGAATATTTGCAAGTCTTTAT 4 9226 9245 1705 560936 N/A N/AGAGGAATATTTGCAAGTCTT 58 9229 9248 57 560937 N/A N/A GTAGAGGAATATTTGCAAGT47 9232 9251 1706 560938 N/A N/A TTGGTAGAGGAATATTTGCA 65 9235 9254 58560939 N/A N/A GTTACATTATTATAGATATT 33 9269 9288 1707 560940 N/A N/ATGTGTTACATTATTATAGAT 20 9272 9291 1708 560941 N/A N/AGAAATGTGTTACATTATTAT 0 9276 9295 1709 560942 N/A N/AACCAGTGAAATGTGTTACAT 56 9282 9301 59 560943 N/A N/A TTCACCAGTGAAATGTGTTA19 9285 9304 1710 560944 N/A N/A TGTTTCACCAGTGAAATGTG 41 9288 9307 1711560945 N/A N/A ACATGTTTCACCAGTGAAAT 0 9291 9310 1712 560946 N/A N/AAAGACATGTTTCACCAGTGA 48 9294 9313 1713 560947 N/A N/AGACAAGACATGTTTCACCAG 28 9297 9316 1714 560948 N/A N/ATATGACAAGACATGTTTCAC 13 9300 9319 1715 560949 N/A N/AGCATATGACAAGACATGTTT 12 9303 9322 1716 560950 N/A N/ATAATGCATATGACAAGACAT 4 9307 9326 1717 560951 N/A N/ACTATAATGCATATGACAAGA 22 9310 9329 1718 560952 N/A N/ATTTCTATAATGCATATGACA 23 9313 9332 1719 560953 N/A N/ATCCTTTCTATAATGCATATG 16 9316 9335 1720 560954 N/A N/ATCTGATTATCCTTTCTATAA 32 9324 9343 1721 560955 N/A N/AAAGTCTGATTATCCTTTCTA 42 9327 9346 1722 560956 N/A N/ATGAAAGTCTGATTATCCTTT 51 9330 9349 60 560957 N/A N/A AACTGAAAGTCTGATTATCC31 9333 9352 1723 560958 N/A N/A TATAACTGAAAGTCTGATTA 6 9336 9355 1724560959 N/A N/A GTTAAAAATATTAATATAAC 3 9350 9369 1725 560960 N/A N/ATGTGCACAAAAATGTTAAAA 0 9363 9382 1726 560961 N/A N/ACTATGTGCACAAAAATGTTA 9 9366 9385 1727 560962 N/A N/ATAGCTATGTGCACAAAAATG 29 9369 9388 1728 560963 N/A N/AAGATAGCTATGTGCACAAAA 41 9372 9391 1729 560964 N/A N/ATGAAGATAGCTATGTGCACA 23 9375 9394 1730 560965 N/A N/ATATTGAAGATAGCTATGTGC 13 9378 9397 1731 560966 N/A N/ATTTTATTGAAGATAGCTATG 4 9381 9400 1732 560967 N/A N/ACAATTTTATTGAAGATAGCT 17 9384 9403 1733 560968 N/A N/AAAACAATTTTATTGAAGATA 27 9387 9406 1734 560969 N/A N/AGTGTATCTTAAAATAATACC 7 9412 9431 1735 560970 N/A N/ATTAGTGTATCTTAAAATAAT 25 9415 9434 1736 560971 N/A N/ATGATCATTTTAGTGTATCTT 34 9423 9442 1737 560972 N/A N/ACCCTTGATCATTTTAGTGTA 7 9427 9446 1738 560973 N/A N/AAATCCCTTGATCATTTTAGT 0 9430 9449 1739 560974 N/A N/ATTGAATCCCTTGATCATTTT 20 9433 9452 1740 560975 N/A N/ATTAGTCTTGAATCCCTTGAT 28 9439 9458 1741 560976 N/A N/ATTGTTTAGTCTTGAATCCCT 40 9443 9462 1742 560977 N/A N/AGAGTTGTTTAGTCTTGAATC 6 9446 9465 1743 560978 N/A N/AATTGAGTTGTTTAGTCTTGA 14 9449 9468 1744 560979 N/A N/ACTAATTGAGTTGTTTAGTCT 0 9452 9471 1745 560980 N/A N/ACAACTAATTGAGTTGTTTAG 0 9455 9474 1746 560981 N/A N/AATTGGTGCAACTAATTGAGT 0 9462 9481 1747 560982 N/A N/ATTTATTGGTGCAACTAATTG 9 9465 9484 1748 560983 N/A N/ATTTTTTATTGGTGCAACTAA 8 9468 9487 1749 560984 N/A N/ATAAGTGTTTTTTATTGGTGC 20 9474 9493 1750 560985 N/A N/AACTGACAGTTTTTTTAAGTG 16 9488 9507 1751 560986 N/A N/AGACACTGACAGTTTTTTTAA 6 9491 9510 1752 560987 N/A N/ATTGGACACTGACAGTTTTTT 0 9494 9513 1753 560988 N/A N/AAGGTTGGACACTGACAGTTT 6 9497 9516 1754 560989 N/A N/ATACAGGTTGGACACTGACAG 0 9500 9519 1755 544120 707 726AGTTCTTGGTGCTCTTGGCT 72 6720 6739 15 337487 804 823 CACTTGTATGTTCACCTCTG80 7389 7408 28 544145 1055 1074 GTTGTCTTTCCAGTCTTCCA 69 9630 9649 16544156 1195 1214 GCTTTGTGATCCCAAGTAGA 61 9770 9789 17 544162 1269 1288GGTTGTTTTCTCCACACTCA 71 10241 10260 18 544166 1353 1372ACCTTCCATTTTGAGACTTC 65 10325 10344 19 544199 1907 1926TACACATACTCTGTGCTGAC 69 10879 10898 20

TABLE 134 Inhibition of ANGPTL3 mRNA by 5-10-5 MOE gapmers targeting SEQID NO: 1 and 2 SEQ SEQ SEQ ID SEQ ID ID NO: NO: ID NO: NO: 2 SEQ ISIS 1Start 1 Stop % 2 Start Stop ID NO Site Site Sequence inhibition SiteSite NO 563720 N/A N/A TATATTGGATAATTTGAAAT 7 11610 11629 1756 563721N/A N/A ATGTATATTGGATAATTTGA 17 11613 11632 1757 563722 N/A N/AGACATGTATATTGGATAATT 20 11616 11635 1758 563723 N/A N/AATGACATGTATATTGGATAA 29 11618 11637 1759 563724 N/A N/ATATATATGACATGTATATTG 9 11623 11642 1760 563725 N/A N/AATGTGACATATAAAAATATA 4 11639 11658 1761 563726 N/A N/AATATGTGACATATAAAAATA 0 11641 11660 1762 563727 N/A N/ATTTATATATGTGACATATAA 0 11646 11665 1763 563728 N/A N/ACTTTTATATATGTGACATAT 16 11648 11667 1764 563729 N/A N/AATCTTTTATATATGTGACAT 13 11650 11669 1765 563730 N/A N/ACATATCTTTTATATATGTGA 2 11653 11672 1766 563731 N/A N/ATCATACATATCTTTTATATA 2 11658 11677 1767 563732 N/A N/ATAGATCATACATATCTTTTA 31 11662 11681 1768 563733 N/A N/ACATAGATCATACATATCTTT 28 11664 11683 1769 563734 N/A N/ACACATAGATCATACATATCT 56 11666 11685 1770 563735 N/A N/AAGGATTCACATAGATCATAC 56 11672 11691 1771 563736 N/A N/ATTAGGATTCACATAGATCAT 24 11674 11693 1772 563737 N/A N/AACTTAGGATTCACATAGATC 49 11676 11695 1773 563738 N/A N/ATTACTTAGGATTCACATAGA 15 11678 11697 1774 563739 N/A N/ATATTTACTTAGGATTCACAT 6 11681 11700 1775 563740 N/A N/AAATATTTACTTAGGATTCAC 28 11683 11702 1776 563741 N/A N/ATGTACTTTTCTGGAACAAAA 63 11701 11720 1777 563742 N/A N/AGATTATTTTTACCTTTATTA 21 11724 11743 1778 563743 N/A N/ATAGATTATTTTTACCTTTAT 5 11726 11745 1779 563744 N/A N/AATTATAGATTATTTTTACCT 12 11730 11749 1780 563745 N/A N/AGAAAATTATAGATTATTTTT 15 11734 11753 1781 563746 N/A N/AGGTCCTGAAAATTATAGATT 7 11740 11759 1782 563747 N/A N/AGTGGTCCTGAAAATTATAGA 29 11742 11761 1783 563748 N/A N/ACTGTGGTCCTGAAAATTATA 37 11744 11763 1784 563749 N/A N/AGTCTGTGGTCCTGAAAATTA 47 11746 11765 1785 563750 N/A N/ATCGACAGCTTAGTCTGTGGT 66 11757 11776 1786 563751 N/A N/ATTTCGACAGCTTAGTCTGTG 41 11759 11778 1787 563752 N/A N/AAATTTCGACAGCTTAGTCTG 40 11761 11780 1788 563753 N/A N/ATTAATTTCGACAGCTTAGTC 35 11763 11782 1789 563754 N/A N/ACGTTAATTTCGACAGCTTAG 50 11765 11784 1790 563755 N/A N/ATGGCCCTAAAAAAATCAGCG 7 11783 11802 1791 563756 N/A N/ATCTGGCCCTAAAAAAATCAG 0 11785 11804 1792 563757 N/A N/ATGGTATTCTGGCCCTAAAAA 37 11791 11810 1793 563758 N/A N/ATTTGGTATTCTGGCCCTAAA 29 11793 11812 1794 563759 N/A N/ACCATTTTGGTATTCTGGCCC 35 11797 11816 1795 563760 N/A N/AGAGGAGCCATTTTGGTATTC 34 11803 11822 1796 563761 N/A N/AGAGAGGAGCCATTTTGGTAT 18 11805 11824 1797 563762 N/A N/AAAGAGAGGAGCCATTTTGGT 17 11807 11826 1798 563763 N/A N/ATGAAATTGTCCAATTTTGGG 28 11829 11848 1799 563764 N/A N/ATTTGAAATTGTCCAATTTTG 10 11831 11850 1800 563765 N/A N/ACATTTGAAATTGTCCAATTT 22 11833 11852 1801 563766 N/A N/ATGCATTTGAAATTGTCCAAT 45 11835 11854 1802 563767 N/A N/AATTTTGCATTTGAAATTGTC 35 11839 11858 1803 563768 N/A N/AATAATGAATTATTTTGCATT 0 11849 11868 1804 563769 N/A N/ATAAATAATGAATTATTTTGC 17 11852 11871 1805 563770 N/A N/ACTCATATATTAAATAATGAA 0 11861 11880 1806 563771 N/A N/AAACTCATATATTAAATAATG 16 11863 11882 1807 563772 N/A N/ATAGAGGAAGCAACTCATATA 7 11873 11892 1808 563773 N/A N/AAATAGAGGAAGCAACTCATA 20 11875 11894 1809 563774 N/A N/ACAAATAGAGGAAGCAACTCA 29 11877 11896 1810 563775 N/A N/AACCAAATAGAGGAAGCAACT 27 11879 11898 1811 563776 N/A N/AAAACCAAATAGAGGAAGCAA 22 11881 11900 1812 563777 N/A N/AGGAAACCAAATAGAGGAAGC 37 11883 11902 1813 563778 N/A N/ATAAGGAAACCAAATAGAGGA 0 11886 11905 1814 563779 N/A N/ATTTAAGGAAACCAAATAGAG 0 11888 11907 1815 563780 N/A N/ATGTTTTCTTCTGGAAGCAGA 5 3100 3119 1816 563781 N/A N/ACTTACTTTAAGTGAAGTTAC 0 3636 3655 1817 563782 N/A N/ATTTTCTACTTACTTTAAGTG 3 3643 3662 1818 563783 N/A N/AACATGAACCCTCTTTATTTT 0 3659 3678 1819 563784 N/A N/AGAAAACATAAACATGAACCC 0 3669 3688 1820 563785 N/A N/AAGATCCACATTGAAAACATA 8 3680 3699 1821 563786 N/A N/ATTAAAAGATCCACATTGAAA 8 3685 3704 1822 563787 N/A N/AGCCTTAGAAATATTTTTTTT 2 3703 3722 1823 563788 N/A N/ACAAATGGCATGCCTTAGAAA 29 3713 3732 1824 563789 N/A N/ATATTTCAAATGGCATGCCTT 24 3718 3737 1825 563790 N/A N/ACAAAGTATTTCAAATGGCAT 8 3723 3742 1826 563791 N/A N/ATGCAACAAAGTATTTCAAAT 0 3728 3747 1827 563792 N/A N/ATCAACAATGCAACAAAGTAT 3 3735 3754 1828 563793 N/A N/AGAAAAAAAAGTATTTCAACA 4 3749 3768 1829 563794 N/A N/AGATTATTTTTCTTGGAAAAA 11 3763 3782 1830 563795 N/A N/AGAAATTTTATTTTCTGGAGA 10 3781 3800 1831 563796 N/A N/AAAATTATAATAGGAAATTTT 14 3793 3812 1832 563797 N/A N/ACTGAATATAATGAATGAAAT 1 7854 7873 1833 563798 N/A N/ATACCTGAATATAATGAATGA 4 7857 7876 1834 563799 N/A N/AGACTACCTGAATATAATGAA 25 7860 7879 1835 563800 N/A N/AATGGACTACCTGAATATAAT 15 7863 7882 1836 563801 N/A N/ATCCATGGACTACCTGAATAT 39 7866 7885 1837 563802 N/A N/AACCATCAAGCCTCCCAAAAC 23 7952 7971 1838 563803 N/A N/ACCTTACCATCAAGCCTCCCA 29 7956 7975 1839 563804 N/A N/AAGTCCCCTTACCATCAAGCC 31 7961 7980 1840 563805 N/A N/ATGTAGTCCCCTTACCATCAA 18 7964 7983 1841 563806 N/A N/AGAATGTAGTCCCCTTACCAT 0 7967 7986 1842 563807 N/A N/AATTGAATGTAGTCCCCTTAC 12 7970 7989 1843 563808 N/A N/AATGATTGAATGTAGTCCCCT 14 7973 7992 1844 563809 N/A N/AGATTAGCAAGTGAATGAATG 13 7990 8009 1845 563810 N/A N/AGTAGATTAGCAAGTGAATGA 25 7993 8012 1846 563811 N/A N/ATTTGTAGATTAGCAAGTGAA 9 7996 8015 1847 563812 N/A N/AATATTTGTAGATTAGCAAGT 0 7999 8018 1848 563813 N/A N/ACCATAAGAGGTTCTCAGTAA 44 8019 8038 1849 563814 N/A N/AGGTCCATAAGAGGTTCTCAG 37 8022 8041 1850 563815 N/A N/ACCTGGTCCATAAGAGGTTCT 25 8025 8044 1851 563816 N/A N/ATAATACCTGGTCCATAAGAG 9 8030 8049 1852 563817 N/A N/ATCCTAATACCTGGTCCATAA 39 8033 8052 1853 563818 N/A N/ATTTTCCTAATACCTGGTCCA 43 8036 8055 1854 563819 N/A N/ATACTTTTCCTAATACCTGGT 43 8039 8058 1855 563820 N/A N/ACGTTACTACTTTTCCTAATA 47 8045 8064 1856 563821 N/A N/AAAGGCTGAGACTGCTTCTCG 46 8067 8086 1857 563822 N/A N/AGATAATAAATTATATGAAGG 5 8083 8102 1858 563823 N/A N/AGTTTGATAATAAATTATATG 0 8087 8106 1859 563824 N/A N/AGTGTAATTGTTTGATAATAA 14 8095 8114 1860 563825 N/A N/AAATGTGTAATTGTTTGATAA 0 8098 8117 1861 563826 N/A N/AGTAATTTACTAACAAATGTG 18 8112 8131 1862 563827 N/A N/AAGTGTAATTTACTAACAAAT 0 8115 8134 1863 563828 N/A N/AATAAGTGTAATTTACTAACA 0 8118 8137 1864 563829 N/A N/AGTAATAAGTGTAATTTACTA 0 8121 8140 1865 563830 N/A N/AGTTGTAATAAGTGTAATTTA 20 8124 8143 1866 563831 N/A N/AACAGTTGTAATAAGTGTAAT 1 8127 8146 1867 563832 N/A N/AATAACAGTTGTAATAAGTGT 4 8130 8149 1868 563833 N/A N/ATTCAAATAATAACAGTTGTA 0 8138 8157 1869 563834 N/A N/AATAATTCAAATAATAACAGT 16 8142 8161 1870 563835 N/A N/AAATTGTGATAAATATAATTC 0 8155 8174 1871 563836 N/A N/AATGTAATTGTGATAAATATA 0 8159 8178 1872 563837 N/A N/AGACATGTAATTGTGATAAAT 8 8162 8181 1873 563838 N/A N/AACAGACATGTAATTGTGATA 33 8165 8184 1874 563839 N/A N/AAGAACAGACATGTAATTGTG 34 8168 8187 1875 563840 N/A N/ATTAAGAACAGACATGTAATT 0 8171 8190 1876 563841 N/A N/AAAGTATATTTAAGAACAGAC 0 8179 8198 1877 563842 N/A N/ATTAAATTGTGATAAGTATAT 1 8191 8210 1878 563843 N/A N/AGAATTAAATTGTGATAAGTA 0 8194 8213 1879 563844 N/A N/AGTGGAATTAAATTGTGATAA 0 8197 8216 1880 563845 N/A N/AGCCGTGGAATTAAATTGTGA 20 8200 8219 1881 563846 N/A N/ATAAGCCGTGGAATTAAATTG 16 8203 8222 1882 563847 N/A N/ATTGTAAGCCGTGGAATTAAA 28 8206 8225 1883 563848 N/A N/ATCATTGTAAGCCGTGGAATT 25 8209 8228 1884 563849 N/A N/ATGATCATTGTAAGCCGTGGA 49 8212 8231 1885 563850 N/A N/ATATAGTTATGATCATTGTAA 0 8220 8239 1886 563851 N/A N/AAATTATAGTTATGATCATTG 0 8223 8242 1887 563852 N/A N/ACTTTAATAATTATAGTTATG 0 8230 8249 1888 563853 N/A N/ATGTCTTTAATAATTATAGTT 4 8233 8252 1889 563854 N/A N/AAATTGTCTTTAATAATTATA 0 8236 8255 1890 563855 N/A N/ATCAAAATTGTCTTTAATAAT 7 8240 8259 1891 563856 N/A N/AATTTAATCAAAATTGTCTTT 0 8246 8265 1892 563857 N/A N/ATAACATTTAATCAAAATTGT 0 8250 8269 1893 563858 N/A N/AACATAACATTTAATCAAAAT 0 8253 8272 1894 563859 N/A N/AATGACATAACATTTAATCAA 13 8256 8275 1895 563860 N/A N/ATACTTATGACATAACATTTA 0 8261 8280 1896 563861 N/A N/ATTACTACTTATGACATAACA 0 8265 8284 1897 563862 N/A N/AAACAGTTACTACTTATGACA 31 8270 8289 1898 563863 N/A N/ATGTAACAGTTACTACTTATG 29 8273 8292 1899 563864 N/A N/ACTTATTTGTAACAGTTACTA 0 8279 8298 1900 563865 N/A N/ATTTCACAGCTTATTTGTAAC 29 8287 8306 1901 563866 N/A N/ATCTTTTCACAGCTTATTTGT 22 8290 8309 1902 563867 N/A N/AGGTTCTTTTCACAGCTTATT 66 8293 8312 1903 563868 N/A N/ACTAGGAGTGGTTCTTTTCAC 37 8301 8320 1904 563869 N/A N/AATGCTAGGAGTGGTTCTTTT 20 8304 8323 1905 563870 N/A N/ACTAATGCTAGGAGTGGTTCT 30 8307 8326 1906 563871 N/A N/AAGAGTGACTAATGCTAGGAG 41 8314 8333 1907 563872 N/A N/AAGAGAATAGAGTGACTAATG 28 8321 8340 1908 563873 N/A N/ATTAATGAGAGAATAGAGTGA 4 8327 8346 1909 563496 608 627CTGTTGGTTTAATTGTTTAT 33 4346 4365 1910 563497 610 629TGCTGTTGGTTTAATTGTTT 29 4348 4367 1911 563498 612 631TATGCTGTTGGTTTAATTGT 27 4350 4369 1912 563499 614 633ACTATGCTGTTGGTTTAATT 24 4352 4371 1913 563500 616 635TGACTATGCTGTTGGTTTAA 68 4354 4373 1914 563501 619 638ATTTGACTATGCTGTTGGTT 45 4357 4376 1915 563502 621 640TTATTTGACTATGCTGTTGG 39 4359 4378 1916 563503 623 642TTTTATTTGACTATGCTGTT 33 4361 4380 1917 563504 625 644TCTTTTATTTGACTATGCTG 55 4363 4382 1918 563505 627 646TTTCTTTTATTTGACTATGC 29 4365 4384 1919 563506 646 665CTTCTGAGCTGATTTTCTAT 40 N/A N/A 1920 563507 648 667 TCCTTCTGAGCTGATTTTCT76 N/A N/A 1921 563508 650 669 AGTCCTTCTGAGCTGATTTT 37 N/A N/A 1922563509 652 671 CTAGTCCTTCTGAGCTGATT 52 N/A N/A 1923 563510 654 673TACTAGTCCTTCTGAGCTGA 52 6667 6686 1924 563511 656 675AATACTAGTCCTTCTGAGCT 41 6669 6688 1925 563512 658 677TGAATACTAGTCCTTCTGAG 55 6671 6690 1926 563513 660 679CTTGAATACTAGTCCTTCTG 43 6673 6692 1927 563514 662 681TTCTTGAATACTAGTCCTTC 34 6675 6694 1928 563515 666 685TGGGTTCTTGAATACTAGTC 52 6679 6698 1929 563516 668 687TGTGGGTTCTTGAATACTAG 34 6681 6700 1930 563517 670 689TCTGTGGGTTCTTGAATACT 43 6683 6702 1931 563518 680 699TAGAGAAATTTCTGTGGGTT 0 6693 6712 1932 563519 684 703AAGATAGAGAAATTTCTGTG 4 6697 6716 1933 563520 686 705GGAAGATAGAGAAATTTCTG 0 6699 6718 1934 563521 694 713CTTGGCTTGGAAGATAGAGA 29 6707 6726 1935 563522 696 715CTCTTGGCTTGGAAGATAGA 51 6709 6728 1936 563523 705 724TTCTTGGTGCTCTTGGCTTG 63 6718 6737 75 544120 707 726 AGTTCTTGGTGCTCTTGGCT86 6720 6739 15 563524 715 734 AAGGGAGTAGTTCTTGGTGC 44 6728 6747 1937563525 716 735 AAAGGGAGTAGTTCTTGGTG 14 6729 6748 1938 563526 717 736GAAAGGGAGTAGTTCTTGGT 33 6730 6749 1939 563527 718 737AGAAAGGGAGTAGTTCTTGG 0 6731 6750 1940 563528 719 738AAGAAAGGGAGTAGTTCTTG 0 6732 6751 1941 563529 720 739GAAGAAAGGGAGTAGTTCTT 0 6733 6752 1942 563530 726 745TCAACTGAAGAAAGGGAGTA 0 6739 6758 1943 337481 728 747ATTCAACTGAAGAAAGGGAG 23 6741 6760 1944 563531 729 748CATTCAACTGAAGAAAGGGA 16 6742 6761 1945 563532 730 749TCATTCAACTGAAGAAAGGG 23 6743 6762 1946 563533 732 751TTTCATTCAACTGAAGAAAG 8 6745 6764 1947 563534 733 752ATTTCATTCAACTGAAGAAA 6 6746 6765 1948 563535 734 753TATTTCATTCAACTGAAGAA 0 6747 6766 1949 563536 735 754TTATTTCATTCAACTGAAGA 0 6748 6767 1950 563537 736 755CTTATTTCATTCAACTGAAG 11 6749 6768 1951 337482 737 756TCTTATTTCATTCAACTGAA 26 6750 6769 1952 563538 738 757TTCTTATTTCATTCAACTGA 17 6751 6770 1953 563539 740 759ATTTCTTATTTCATTCAACT 18 6753 6772 1954 563540 743 762TACATTTCTTATTTCATTCA 20 6756 6775 1955 563541 767 786TTCAGCAGGAATGCCATCAT 34 N/A N/A 1956 563542 768 787 ATTCAGCAGGAATGCCATCA2 N/A N/A 1957 563543 769 788 CATTCAGCAGGAATGCCATC 21 N/A N/A 1958563544 770 789 ACATTCAGCAGGAATGCCAT 5 N/A N/A 1959 563545 771 790TACATTCAGCAGGAATGCCA 37 N/A N/A 1960 563546 772 791 GTACATTCAGCAGGAATGCC50 7357 7376 1961 563547 773 792 GGTACATTCAGCAGGAATGC 64 7358 7377 76563548 774 793 TGGTACATTCAGCAGGAATG 42 7359 7378 1962 563549 775 794GTGGTACATTCAGCAGGAAT 51 7360 7379 1963 563550 776 795GGTGGTACATTCAGCAGGAA 24 7361 7380 1964 563551 777 796TGGTGGTACATTCAGCAGGA 47 7362 7381 1965 563552 778 797ATGGTGGTACATTCAGCAGG 0 7363 7382 1966 563553 779 798AATGGTGGTACATTCAGCAG 15 7364 7383 1967 563554 780 799AAATGGTGGTACATTCAGCA 32 7365 7384 1968 563555 781 800TAAATGGTGGTACATTCAGC 29 7366 7385 1969 563556 783 802TATAAATGGTGGTACATTCA 33 7368 7387 1970 563557 784 803TTATAAATGGTGGTACATTC 1 7369 7388 1971 563558 785 804GTTATAAATGGTGGTACATT 4 7370 7389 1972 563559 786 805TGTTATAAATGGTGGTACAT 0 7371 7390 1973 563560 787 806CTGTTATAAATGGTGGTACA 4 7372 7391 1974 563561 788 807TCTGTTATAAATGGTGGTAC 29 7373 7392 1975 337484 789 808CTCTGTTATAAATGGTGGTA 62 7374 7393 74 563562 792 811 CACCTCTGTTATAAATGGTG22 7377 7396 1976 563563 793 812 TCACCTCTGTTATAAATGGT 38 7378 7397 1977337485 794 813 TTCACCTCTGTTATAAATGG 18 7379 7398 1978 563564 795 814GTTCACCTCTGTTATAAATG 52 7380 7399 1979 563565 797 816ATGTTCACCTCTGTTATAAA 24 7382 7401 1980 563566 798 817TATGTTCACCTCTGTTATAA 2 7383 7402 1981 337486 799 818GTATGTTCACCTCTGTTATA 32 7384 7403 1982 563567 800 819TGTATGTTCACCTCTGTTAT 38 7385 7404 1983 337487 804 823CACTTGTATGTTCACCTCTG 87 7389 7408 28 563568 1128 1147TAATCGCAACTAGATGTAGC 39 9703 9722 1984 563569 1129 1148GTAATCGCAACTAGATGTAG 26 9704 9723 1985 563570 1130 1149AGTAATCGCAACTAGATGTA 17 9705 9724 1986 563571 1131 1150CAGTAATCGCAACTAGATGT 43 9706 9725 1987 563572 1132 1151CCAGTAATCGCAACTAGATG 39 9707 9726 1988 563573 1133 1152GCCAGTAATCGCAACTAGAT 59 9708 9727 1989 563574 1134 1153TGCCAGTAATCGCAACTAGA 57 9709 9728 1990 563575 1135 1154TTGCCAGTAATCGCAACTAG 54 9710 9729 1991 563576 1136 1155ATTGCCAGTAATCGCAACTA 43 9711 9730 1992 563577 1137 1156CATTGCCAGTAATCGCAACT 49 9712 9731 1993 563578 1138 1157ACATTGCCAGTAATCGCAAC 59 9713 9732 1994 563579 1139 1158GACATTGCCAGTAATCGCAA 64 9714 9733 1995 563580 1140 1159GGACATTGCCAGTAATCGCA 79 9715 9734 77 563581 1141 1160GGGACATTGCCAGTAATCGC 47 9716 9735 1996 563582 1162 1181TTGTTTTCCGGGATTGCATT 20 9737 9756 1997 563583 1163 1182TTTGTTTTCCGGGATTGCAT 31 9738 9757 1998 563584 1167 1186AATCTTTGTTTTCCGGGATT 14 9742 9761 1999 563585 1168 1187AAATCTTTGTTTTCCGGGAT 54 9743 9762 2000 563586 1175 1194AAACACCAAATCTTTGTTTT 32 9750 9769 2001 563587 1176 1195AAAACACCAAATCTTTGTTT 7 9751 9770 2002 563588 1180 1199GTAGAAAACACCAAATCTTT 18 9755 9774 2003 563589 1181 1200AGTAGAAAACACCAAATCTT 0 9756 9775 2004 563590 1185 1204CCCAAGTAGAAAACACCAAA 26 9760 9779 2005 563591 1186 1205TCCCAAGTAGAAAACACCAA 27 9761 9780 2006 563592 1190 1209GTGATCCCAAGTAGAAAACA 26 9765 9784 2007 563593 1191 1210TGTGATCCCAAGTAGAAAAC 28 9766 9785 2008 563594 1192 1211TTGTGATCCCAAGTAGAAAA 12 9767 9786 2009 563595 1193 1212TTTGTGATCCCAAGTAGAAA 14 9768 9787 2010 563596 1200 1219CTTTTGCTTTGTGATCCCAA 64 9775 9794 2011 563597 1204 1223TGTCCTTTTGCTTTGTGATC 24 9779 9798 2012 563598 1205 1224GTGTCCTTTTGCTTTGTGAT 31 9780 9799 2013 563599 1206 1225AGTGTCCTTTTGCTTTGTGA 41 9781 9800 2014 563600 1210 1229TTGAAGTGTCCTTTTGCTTT 21 9785 9804 2015 563601 1211 1230GTTGAAGTGTCCTTTTGCTT 35 9786 9805 2016 563602 1212 1231AGTTGAAGTGTCCTTTTGCT 27 9787 9806 2017 563603 1213 1232CAGTTGAAGTGTCCTTTTGC 17 9788 9807 2018 563604 1214 1233ACAGTTGAAGTGTCCTTTTG 0 9789 9808 2019 563605 1215 1234GACAGTTGAAGTGTCCTTTT 19 9790 9809 2020 563606 1216 1235GGACAGTTGAAGTGTCCTTT 34 9791 9810 2021 563607 1217 1236TGGACAGTTGAAGTGTCCTT 12 9792 9811 2022 563608 1218 1237CTGGACAGTTGAAGTGTCCT 39 9793 9812 2023 563609 1219 1238TCTGGACAGTTGAAGTGTCC 10 9794 9813 2024 563610 1220 1239CTCTGGACAGTTGAAGTGTC 6 9795 9814 2025 563611 1221 1240CCTCTGGACAGTTGAAGTGT 24 9796 9815 2026 563612 1222 1241CCCTCTGGACAGTTGAAGTG 24 9797 9816 2027 563613 1223 1242ACCCTCTGGACAGTTGAAGT 31 9798 9817 2028 563614 1224 1243AACCCTCTGGACAGTTGAAG 34 9799 9818 2029 563615 1225 1244TAACCCTCTGGACAGTTGAA 34 9800 9819 2030 563616 1226 1245ATAACCCTCTGGACAGTTGA 31 9801 9820 2031 563617 1227 1246AATAACCCTCTGGACAGTTG 22 9802 9821 2032 563618 1228 1247GAATAACCCTCTGGACAGTT 25 9803 9822 2033 563619 1229 1248TGAATAACCCTCTGGACAGT 18 9804 9823 2034 563620 1230 1249CTGAATAACCCTCTGGACAG 24 9805 9824 2035 563621 1231 1250CCTGAATAACCCTCTGGACA 39 9806 9825 2036 563622 1232 1251TCCTGAATAACCCTCTGGAC 31 N/A N/A 2037 563623 1233 1252CTCCTGAATAACCCTCTGGA 15 N/A N/A 2038 563624 1234 1253CCTCCTGAATAACCCTCTGG 27 N/A N/A 2039 563625 1235 1254GCCTCCTGAATAACCCTCTG 25 N/A N/A 2040 563626 1236 1255AGCCTCCTGAATAACCCTCT 32 N/A N/A 2041 563627 1237 1256CAGCCTCCTGAATAACCCTC 44 N/A N/A 2042 563628 1238 1257CCAGCCTCCTGAATAACCCT 26 N/A N/A 2043 563629 1239 1258ACCAGCCTCCTGAATAACCC 23 N/A N/A 2044 337503 1240 1259CACCAGCCTCCTGAATAACC 25 N/A N/A 2045 563630 1241 1260CCACCAGCCTCCTGAATAAC 26 N/A N/A 2046 563631 1242 1261ACCACCAGCCTCCTGAATAA 25 N/A N/A 2047 563632 1243 1262CACCACCAGCCTCCTGAATA 33 N/A N/A 2048 563633 1244 1263CCACCACCAGCCTCCTGAAT 45 N/A N/A 2049 563634 1248 1267CATGCCACCACCAGCCTCCT 54 10220 10239 2050 563635 1250 1269ATCATGCCACCACCAGCCTC 58 10222 10241 2051 563636 1251 1270CATCATGCCACCACCAGCCT 61 10223 10242 2052 563637 1255 1274CACTCATCATGCCACCACCA 68 10227 10246 78 563638 1256 1275ACACTCATCATGCCACCACC 65 10228 10247 2053 563639 1260 1279CTCCACACTCATCATGCCAC 76 10232 10251 79 563640 1262 1281TTCTCCACACTCATCATGCC 55 10234 10253 2054 563641 1263 1282TTTCTCCACACTCATCATGC 63 10235 10254 80 563642 1264 1283TTTTCTCCACACTCATCATG 24 10236 10255 2055 563643 1265 1284GTTTTCTCCACACTCATCAT 53 10237 10256 2056 563644 1857 1876ATTTAAGAACTGTACAATTA 7 10829 10848 2057 563645 1858 1877CATTTAAGAACTGTACAATT 15 10830 10849 2058 563646 1859 1878ACATTTAAGAACTGTACAAT 4 10831 10850 2059 563647 1860 1879AACATTTAAGAACTGTACAA 4 10832 10851 2060 563648 1861 1880CAACATTTAAGAACTGTACA 4 10833 10852 2061 563649 1862 1881ACAACATTTAAGAACTGTAC 22 10834 10853 2062 563650 1863 1882TACAACATTTAAGAACTGTA 21 10835 10854 2063 563651 1864 1883CTACAACATTTAAGAACTGT 44 10836 10855 2064 563652 1865 1884ACTACAACATTTAAGAACTG 20 10837 10856 2065 563653 1866 1885TACTACAACATTTAAGAACT 15 10838 10857 2066 563654 1867 1886ATACTACAACATTTAAGAAC 17 10839 10858 2067 563655 1868 1887AATACTACAACATTTAAGAA 11 10840 10859 2068 563656 1869 1888TAATACTACAACATTTAAGA 9 10841 10860 2069 563657 1870 1889TTAATACTACAACATTTAAG 3 10842 10861 2070 563658 1874 1893GAAATTAATACTACAACATT 0 10846 10865 2071 563659 1878 1897TTTTGAAATTAATACTACAA 0 10850 10869 2072 563660 1879 1898GTTTTGAAATTAATACTACA 15 10851 10870 2073 563661 1880 1899AGTTTTGAAATTAATACTAC 2 10852 10871 2074 563662 1881 1900TAGTTTTGAAATTAATACTA 14 10853 10872 2075 563663 1882 1901TTAGTTTTGAAATTAATACT 8 10854 10873 2076 563664 1888 1907CGATTTTTAGTTTTGAAATT 0 10860 10879 2077 563665 1889 1908ACGATTTTTAGTTTTGAAAT 0 10861 10880 2078 563666 1890 1909GACGATTTTTAGTTTTGAAA 20 10862 10881 2079 563667 1891 1910TGACGATTTTTAGTTTTGAA 17 10863 10882 2080 563668 1892 1911CTGACGATTTTTAGTTTTGA 64 10864 10883 2081 563669 1893 1912GCTGACGATTTTTAGTTTTG 66 10865 10884 81 563670 1894 1913TGCTGACGATTTTTAGTTTT 45 10866 10885 2082 563671 1895 1914GTGCTGACGATTTTTAGTTT 42 10867 10886 2083 563672 1896 1915TGTGCTGACGATTTTTAGTT 50 10868 10887 2084 563673 1897 1916CTGTGCTGACGATTTTTAGT 55 10869 10888 2085 563674 1898 1917TCTGTGCTGACGATTTTTAG 53 10870 10889 2086 563675 1899 1918CTCTGTGCTGACGATTTTTA 49 10871 10890 2087 563676 1900 1919ACTCTGTGCTGACGATTTTT 22 10872 10891 2088 563677 1901 1920TACTCTGTGCTGACGATTTT 8 10873 10892 2089 563678 1902 1921ATACTCTGTGCTGACGATTT 61 10874 10893 2090 563679 1903 1922CATACTCTGTGCTGACGATT 68 10875 10894 2091 563680 1904 1923ACATACTCTGTGCTGACGAT 4 10876 10895 2092 563681 1905 1924CACATACTCTGTGCTGACGA 73 10877 10896 82 563682 1909 1928TTTACACATACTCTGTGCTG 67 10881 10900 83 563683 1911 1930TTTTTACACATACTCTGTGC 58 10883 10902 2093 563684 1915 1934CAGATTTTTACACATACTCT 54 10887 10906 2094 563685 1916 1935ACAGATTTTTACACATACTC 52 10888 10907 2095 563686 1917 1936TACAGATTTTTACACATACT 40 10889 10908 2096 563687 1918 1937TTACAGATTTTTACACATAC 22 10890 10909 2097 337528 1920 1939TATTACAGATTTTTACACAT 4 6720 6739 2098 563688 1922 1941TGTATTACAGATTTTTACAC 0 10894 10913 2099 563689 1935 1954CAGTTTAAAAATTTGTATTA 8 10907 10926 2100 563690 1938 1957CATCAGTTTAAAAATTTGTA 18 10910 10929 2101 563691 1941 1960AAGCATCAGTTTAAAAATTT 16 10913 10932 2102 563692 1942 1961GAAGCATCAGTTTAAAAATT 16 10914 10933 2103 563693 1951 1970TAGCAAAATGAAGCATCAGT 40 10923 10942 2104 563694 1952 1971GTAGCAAAATGAAGCATCAG 42 10924 10943 2105 563695 1953 1972TGTAGCAAAATGAAGCATCA 44 10925 10944 2106 563696 1954 1973TTGTAGCAAAATGAAGCATC 48 10926 10945 2107 563697 1955 1974TTTGTAGCAAAATGAAGCAT 19 10927 10946 2108 563698 1974 1993AACATTTACTCCAAATTATT 27 10946 10965 2109 563699 1976 1995CAAACATTTACTCCAAATTA 23 10948 10967 2110 563700 1978 1997ATCAAACATTTACTCCAAAT 24 10950 10969 2111 563701 1981 2000CATATCAAACATTTACTCCA 61 10953 10972 2112 563702 1982 2001TCATATCAAACATTTACTCC 50 10954 10973 2113 563703 1983 2002ATCATATCAAACATTTACTC 31 10955 10974 2114 563704 1990 2009TAAATAAATCATATCAAACA 10 10962 10981 2115 563705 1993 2012TCATAAATAAATCATATCAA 20 10965 10984 2116 563706 1994 2013TTCATAAATAAATCATATCA 11 10966 10985 2117 563707 1995 2014TTTCATAAATAAATCATATC 5 10967 10986 2118 563708 1996 2015GTTTCATAAATAAATCATAT 0 10968 10987 2119 563709 1997 2016GGTTTCATAAATAAATCATA 8 10969 10988 2120 563710 1998 2017AGGTTTCATAAATAAATCAT 15 10970 10989 2121 563711 1999 2018TAGGTTTCATAAATAAATCA 19 10971 10990 2122 563712 2001 2020ATTAGGTTTCATAAATAAAT 12 10973 10992 2123 563713 2002 2021CATTAGGTTTCATAAATAAA 2 10974 10993 2124 563714 2003 2022TCATTAGGTTTCATAAATAA 7 10975 10994 2125 563715 2004 2023TTCATTAGGTTTCATAAATA 11 10976 10995 2126 563716 2005 2024CTTCATTAGGTTTCATAAAT 15 10977 10996 2127 563717 2006 2025GCTTCATTAGGTTTCATAAA 49 10978 10997 2128 563718 2010 2029TTCTGCTTCATTAGGTTTCA 57 10982 11001 2129 563719 2013 2032TAATTCTGCTTCATTAGGTT 43 10985 11004 2130

TABLE 135 Inhibition of ANGPTL3 mRNA by 5-10-5 MOE gapmers targeting SEQID NO: 1 and 2 SEQ SEQ SEQ ID SEQ ID ID NO: NO: ID NO: NO: 2 SEQ ISIS 1Start 1 Stop % 2 Start Stop ID NO Site Site Sequence inhibition SiteSite NO 566915 343 362 TATGTAGTTCTTCTCAGTTC 22 3447 3466 2131 566916 350369 TAGTTTATATGTAGTTCTTC 21 3454 3473 2132 566917 354 373CTTGTAGTTTATATGTAGTT 12 3458 3477 2133 566918 358 377TTGACTTGTAGTTTATATGT 12 3462 3481 2134 566919 360 379TTTTGACTTGTAGTTTATAT 0 3464 3483 2135 566920 362 381ATTTTTGACTTGTAGTTTAT 7 3466 3485 2136 566921 367 386TCTTCATTTTTGACTTGTAG 33 3471 3490 2137 566922 371 390TACCTCTTCATTTTTGACTT 22 3475 3494 2138 566923 377 396ATTCTTTACCTCTTCATTTT 12 3481 3500 2139 566924 387 406CAAGTGACATATTCTTTACC 36 3491 3510 2140 566925 389 408TTCAAGTGACATATTCTTTA 31 3493 3512 2141 566926 394 413TTGAGTTCAAGTGACATATT 18 3498 3517 2142 566927 396 415AGTTGAGTTCAAGTGACATA 6 3500 3519 2143 566928 400 419TTTGAGTTGAGTTCAAGTGA 11 3504 3523 2144 566929 408 427TTTCAAGTTTTGAGTTGAGT 15 3512 3531 2145 566930 410 429GCTTTCAAGTTTTGAGTTGA 13 3514 3533 2146 566931 412 431AGGCTTTCAAGTTTTGAGTT 22 3516 3535 2147 566932 416 435TAGGAGGCTTTCAAGTTTTG 4 3520 3539 2148 566933 419 438TTCTAGGAGGCTTTCAAGTT 35 3523 3542 2149 566934 421 440TCTTCTAGGAGGCTTTCAAG 26 3525 3544 2150 566935 429 448GAATTTTTTCTTCTAGGAGG 1 3533 3552 2151 566936 434 453AAGTAGAATTTTTTCTTCTA 0 3538 3557 2152 566937 436 455TGAAGTAGAATTTTTTCTTC 11 3540 3559 2153 566938 438 457GTTGAAGTAGAATTTTTTCT 29 3542 3561 2154 566939 441 460TTTGTTGAAGTAGAATTTTT 11 3545 3564 2155 566940 443 462TTTTTGTTGAAGTAGAATTT 35 3547 3566 2156 566941 464 483TTGCTCTTCTAAATATTTCA 35 3568 3587 2157 566942 466 485AGTTGCTCTTCTAAATATTT 53 3570 3589 2158 566943 468 487TTAGTTGCTCTTCTAAATAT 18 3572 3591 2159 566944 471 490TAGTTAGTTGCTCTTCTAAA 38 3575 3594 2160 566945 476 495TAAGTTAGTTAGTTGCTCTT 28 3580 3599 2161 566946 478 497ATTAAGTTAGTTAGTTGCTC 28 3582 3601 2162 566947 480 499GAATTAAGTTAGTTAGTTGC 27 3584 3603 2163 566948 482 501TTGAATTAAGTTAGTTAGTT 21 3586 3605 2164 566949 484 503TTTTGAATTAAGTTAGTTAG 2 3588 3607 2165 566950 487 506TGATTTTGAATTAAGTTAGT 9 3591 3610 2166 566951 490 509GGTTGATTTTGAATTAAGTT 52 3594 3613 2167 566952 497 516AGTTTCAGGTTGATTTTGAA 13 3601 3620 2168 566953 501 520CTGGAGTTTCAGGTTGATTT 50 3605 3624 2169 566954 507 526GGTGTTCTGGAGTTTCAGGT 35 3611 3630 2170 566955 509 528TGGGTGTTCTGGAGTTTCAG 18 3613 3632 2171 566956 511 530TCTGGGTGTTCTGGAGTTTC 32 3615 3634 2172 566957 513 532CTTCTGGGTGTTCTGGAGTT 28 3617 3636 2173 566958 515 534TACTTCTGGGTGTTCTGGAG 23 3619 3638 2174 566959 517 536GTTACTTCTGGGTGTTCTGG 12 3621 3640 2175 566960 519 538AAGTTACTTCTGGGTGTTCT 1 3623 3642 2176 566961 522 541GTGAAGTTACTTCTGGGTGT 0 3626 3645 2177 566962 528 547TTTTAAGTGAAGTTACTTCT 6 N/A N/A 2178 566963 530 549 AGTTTTAAGTGAAGTTACTT16 N/A N/A 2179 566964 532 551 AAAGTTTTAAGTGAAGTTAC 12 N/A N/A 2180566965 535 554 ACAAAAGTTTTAAGTGAAGT 8 N/A N/A 2181 337474 537 556CTACAAAAGTTTTAAGTGAA 10 N/A N/A 2182 566966 539 558 TTCTACAAAAGTTTTAAGTG46 N/A N/A 2183 566967 544 563 TGTTTTTCTACAAAAGTTTT 12 N/A N/A 2184566968 546 565 CTTGTTTTTCTACAAAAGTT 0 N/A N/A 2185 566969 552 571TATTATCTTGTTTTTCTACA 0 4290 4309 2186 566970 557 576GATGCTATTATCTTGTTTTT 18 4295 4314 2187 566971 560 579TTTGATGCTATTATCTTGTT 22 4298 4317 2188 566972 562 581TCTTTGATGCTATTATCTTG 21 4300 4319 2189 566973 569 588GAGAAGGTCTTTGATGCTAT 37 4307 4326 2190 566974 574 593GTCTGGAGAAGGTCTTTGAT 26 4312 4331 2191 566975 576 595CGGTCTGGAGAAGGTCTTTG 20 4314 4333 2192 566976 578 597CACGGTCTGGAGAAGGTCTT 53 4316 4335 2193 566977 580 599TCCACGGTCTGGAGAAGGTC 58 4318 4337 2194 566978 582 601CTTCCACGGTCTGGAGAAGG 39 4320 4339 2195 566979 584 603GTCTTCCACGGTCTGGAGAA 63 4322 4341 2196 566980 586 605TGGTCTTCCACGGTCTGGAG 81 4324 4343 2197 566981 588 607ATTGGTCTTCCACGGTCTGG 57 4326 4345 2198 566982 590 609ATATTGGTCTTCCACGGTCT 60 4328 4347 2199 566983 592 611TTATATTGGTCTTCCACGGT 49 4330 4349 2200 566984 594 613GTTTATATTGGTCTTCCACG 54 4332 4351 2201 566985 596 615TTGTTTATATTGGTCTTCCA 36 4334 4353 2202 566986 598 617AATTGTTTATATTGGTCTTC 23 4336 4355 2203 566987 600 619TTAATTGTTTATATTGGTCT 26 4338 4357 2204 566988 602 621GTTTAATTGTTTATATTGGT 23 4340 4359 2205 566989 604 623TGGTTTAATTGTTTATATTG 8 4342 4361 2206 566990 606 625GTTGGTTTAATTGTTTATAT 1 4344 4363 2207 544120 707 726AGTTCTTGGTGCTCTTGGCT 78 6720 6739 15 337487 804 823 CACTTGTATGTTCACCTCTG82 7389 7408 28 566991 912 931 TTTGTGATCCATCTATTCGA 25 7899 7918 2208566992 913 932 TTTTGTGATCCATCTATTCG 12 7900 7919 2209 566993 920 939ATTGAAGTTTTGTGATCCAT 32 7907 7926 2210 566994 921 940CATTGAAGTTTTGTGATCCA 26 7908 7927 2211 566995 922 941TCATTGAAGTTTTGTGATCC 0 7909 7928 2212 566996 923 942TTCATTGAAGTTTTGTGATC 1 7910 7929 2213 566997 924 943TTTCATTGAAGTTTTGTGAT 20 7911 7930 2214 566998 944 963ATATTTGTAGTTCTCCCACG 35 7931 7950 2215 566999 952 971CCAAAACCATATTTGTAGTT 13 7939 7958 2216 567000 953 972CCCAAAACCATATTTGTAGT 21 7940 7959 2217 567001 954 973TCCCAAAACCATATTTGTAG 0 7941 7960 2218 567002 955 974CTCCCAAAACCATATTTGTA 5 7942 7961 2219 567003 958 977AGCCTCCCAAAACCATATTT 0 7945 7964 2220 567004 960 979CAAGCCTCCCAAAACCATAT 14 7947 7966 2221 567005 961 980TCAAGCCTCCCAAAACCATA 0 7948 7967 2222 567006 962 981ATCAAGCCTCCCAAAACCAT 17 7949 7968 2223 567007 963 982CATCAAGCCTCCCAAAACCA 31 7950 7969 2224 567008 964 983CCATCAAGCCTCCCAAAACC 11 7951 7970 2225 567009 965 984TCCATCAAGCCTCCCAAAAC 27 N/A N/A 2226 567010 966 985 CTCCATCAAGCCTCCCAAAA42 N/A N/A 2227 567011 972 991 AAAATTCTCCATCAAGCCTC 48 N/A N/A 2228567012 974 993 CCAAAATTCTCCATCAAGCC 41 N/A N/A 2229 567013 975 994ACCAAAATTCTCCATCAAGC 49 N/A N/A 2230 567014 978 997 CCAACCAAAATTCTCCATCA32 N/A N/A 2231 567015 979 998 CCCAACCAAAATTCTCCATC 47 N/A N/A 2232337497 980 999 GCCCAACCAAAATTCTCCAT 46 N/A N/A 2233 567016 981 1000GGCCCAACCAAAATTCTCCA 48 N/A N/A 2234 567017 982 1001AGGCCCAACCAAAATTCTCC 30 9557 9576 2235 567018 983 1002TAGGCCCAACCAAAATTCTC 0 9558 9577 2236 567019 984 1003CTAGGCCCAACCAAAATTCT 31 9559 9578 2237 567020 985 1004TCTAGGCCCAACCAAAATTC 39 9560 9579 2238 233721 986 1005CTCTAGGCCCAACCAAAATT 15 9561 9580 2239 567021 987 1006TCTCTAGGCCCAACCAAAAT 36 9562 9581 2240 567022 988 1007TTCTCTAGGCCCAACCAAAA 26 9563 9582 2241 567023 989 1008CTTCTCTAGGCCCAACCAAA 44 9564 9583 2242 567024 993 1012ATATCTTCTCTAGGCCCAAC 29 9568 9587 2243 567025 994 1013TATATCTTCTCTAGGCCCAA 41 9569 9588 2244 567026 995 1014GTATATCTTCTCTAGGCCCA 53 9570 9589 2245 567027 1000 1019ATGGAGTATATCTTCTCTAG 18 9575 9594 2246 567028 1004 1023CACTATGGAGTATATCTTCT 35 9579 9598 2247 567029 1005 1024TCACTATGGAGTATATCTTC 9 9580 9599 2248 567030 1006 1025TTCACTATGGAGTATATCTT 11 9581 9600 2249 567031 1010 1029TTGCTTCACTATGGAGTATA 43 9585 9604 2250 567032 1011 1030ATTGCTTCACTATGGAGTAT 4 9586 9605 2251 567033 1015 1034TTAGATTGCTTCACTATGGA 17 9590 9609 2252 567034 1016 1035ATTAGATTGCTTCACTATGG 35 9591 9610 2253 567035 1017 1036AATTAGATTGCTTCACTATG 18 9592 9611 2254 567036 1018 1037TAATTAGATTGCTTCACTAT 17 9593 9612 2255 567037 1019 1038ATAATTAGATTGCTTCACTA 19 9594 9613 2256 567038 1020 1039CATAATTAGATTGCTTCACT 27 9595 9614 2257 567039 1021 1040ACATAATTAGATTGCTTCAC 17 9596 9615 2258 337498 1022 1041AACATAATTAGATTGCTTCA 9 9597 9616 2259 567040 1023 1042AAACATAATTAGATTGCTTC 0 9598 9617 2260 567041 1024 1043AAAACATAATTAGATTGCTT 0 9599 9618 2261 567042 1025 1044TAAAACATAATTAGATTGCT 23 9600 9619 2262 567043 1026 1045GTAAAACATAATTAGATTGC 25 9601 9620 2263 567044 1027 1046CGTAAAACATAATTAGATTG 0 9602 9621 2264 567045 1048 1067TTCCAGTCTTCCAACTCAAT 9 9623 9642 2265 337500 1050 1069CTTTCCAGTCTTCCAACTCA 30 9625 9644 2266 567046 1057 1076TTGTTGTCTTTCCAGTCTTC 40 9632 9651 2267 567047 1064 1083ATAATGTTTGTTGTCTTTCC 26 9639 9658 2268 567048 1065 1084TATAATGTTTGTTGTCTTTC 6 9640 9659 2269 567049 1066 1085ATATAATGTTTGTTGTCTTT 9 9641 9660 2270 567050 1069 1088TCAATATAATGTTTGTTGTC 20 9644 9663 2271 567051 1073 1092ATATTCAATATAATGTTTGT 15 9648 9667 2272 567052 1074 1093AATATTCAATATAATGTTTG 16 9649 9668 2273 567053 1075 1094GAATATTCAATATAATGTTT 7 9650 9669 2274 567054 1076 1095AGAATATTCAATATAATGTT 3 9651 9670 2275 567055 1077 1096AAGAATATTCAATATAATGT 7 9652 9671 2276 567056 1085 1104CAAGTAAAAAGAATATTCAA 0 9660 9679 2277 567057 1086 1105CCAAGTAAAAAGAATATTCA 0 9661 9680 2278 567058 1087 1106CCCAAGTAAAAAGAATATTC 13 9662 9681 2279 567059 1090 1109TTTCCCAAGTAAAAAGAATA 0 9665 9684 2280 567060 1091 1110ATTTCCCAAGTAAAAAGAAT 2 9666 9685 2281 567061 1092 1111GATTTCCCAAGTAAAAAGAA 14 9667 9686 2282 567062 1093 1112TGATTTCCCAAGTAAAAAGA 14 9668 9687 2283 567063 1127 1146AATCGCAACTAGATGTAGCG 15 9702 9721 2284 563874 1586 1605ATTCTTTAAGGTTATGTGAT 13 10558 10577 2285 563875 1587 1606TATTCTTTAAGGTTATGTGA 25 10559 10578 2286 563876 1591 1610ACGGTATTCTTTAAGGTTAT 50 10563 10582 2287 563877 1592 1611AACGGTATTCTTTAAGGTTA 48 10564 10583 2288 563878 1593 1612AAACGGTATTCTTTAAGGTT 45 10565 10584 2289 563879 1594 1613TAAACGGTATTCTTTAAGGT 16 10566 10585 2290 563880 1595 1614GTAAACGGTATTCTTTAAGG 14 10567 10586 2291 563881 1596 1615TGTAAACGGTATTCTTTAAG 0 10568 10587 2292 563882 1597 1616ATGTAAACGGTATTCTTTAA 10 10569 10588 2293 563883 1598 1617AATGTAAACGGTATTCTTTA 12 10570 10589 2294 563884 1599 1618AAATGTAAACGGTATTCTTT 15 10571 10590 2295 563885 1600 1619GAAATGTAAACGGTATTCTT 13 10572 10591 2296 563886 1601 1620AGAAATGTAAACGGTATTCT 22 10573 10592 2297 563887 1602 1621GAGAAATGTAAACGGTATTC 35 10574 10593 2298 563888 1603 1622TGAGAAATGTAAACGGTATT 14 10575 10594 2299 563889 1604 1623TTGAGAAATGTAAACGGTAT 0 10576 10595 2300 563890 1605 1624ATTGAGAAATGTAAACGGTA 18 10577 10596 2301 563891 1606 1625GATTGAGAAATGTAAACGGT 40 10578 10597 2302 563892 1607 1626TGATTGAGAAATGTAAACGG 33 10579 10598 2303 563893 1608 1627TTGATTGAGAAATGTAAACG 7 10580 10599 2304 563894 1609 1628TTTGATTGAGAAATGTAAAC 0 10581 10600 2305 563895 1610 1629TTTTGATTGAGAAATGTAAA 0 10582 10601 2306 563896 1611 1630ATTTTGATTGAGAAATGTAA 0 10583 10602 2307 563897 1612 1631AATTTTGATTGAGAAATGTA 0 10584 10603 2308 563898 1613 1632GAATTTTGATTGAGAAATGT 4 10585 10604 2309 563899 1614 1633AGAATTTTGATTGAGAAATG 4 10586 10605 2310 563900 1615 1634AAGAATTTTGATTGAGAAAT 26 10587 10606 2311 563901 1617 1636ATAAGAATTTTGATTGAGAA 4 10589 10608 2312 563902 1618 1637TATAAGAATTTTGATTGAGA 0 10590 10609 2313 563903 1619 1638TTATAAGAATTTTGATTGAG 0 10591 10610 2314 563904 1620 1639ATTATAAGAATTTTGATTGA 0 10592 10611 2315 563905 1621 1640TATTATAAGAATTTTGATTG 3 10593 10612 2316 563906 1622 1641GTATTATAAGAATTTTGATT 1 10594 10613 2317 563907 1623 1642AGTATTATAAGAATTTTGAT 44 10595 10614 2318 563908 1624 1643TAGTATTATAAGAATTTTGA 29 10596 10615 2319 563909 1632 1651AAAACAAATAGTATTATAAG 11 10604 10623 2320 563910 1633 1652TAAAACAAATAGTATTATAA 16 10605 10624 2321 563911 1652 1671ATTCCCACATCACAAAATTT 27 10624 10643 2322 563912 1653 1672GATTCCCACATCACAAAATT 21 10625 10644 2323 563913 1654 1673TGATTCCCACATCACAAAAT 49 10626 10645 2324 563914 1658 1677AAATTGATTCCCACATCACA 47 10630 10649 2325 563915 1659 1678AAAATTGATTCCCACATCAC 48 10631 10650 2326 563916 1663 1682ATCTAAAATTGATTCCCACA 58 10635 10654 2327 563917 1667 1686GACCATCTAAAATTGATTCC 41 10639 10658 2328 563918 1668 1687TGACCATCTAAAATTGATTC 25 10640 10659 2329 563919 1669 1688GTGACCATCTAAAATTGATT 33 10641 10660 2330 563920 1670 1689TGTGACCATCTAAAATTGAT 34 10642 10661 2331 563921 1671 1690TTGTGACCATCTAAAATTGA 20 10643 10662 2332 563922 1672 1691ATTGTGACCATCTAAAATTG 2 10644 10663 2333 563923 1673 1692GATTGTGACCATCTAAAATT 43 10645 10664 2334 563924 1674 1693AGATTGTGACCATCTAAAAT 39 10646 10665 2335 563925 1675 1694TAGATTGTGACCATCTAAAA 36 10647 10666 2336 563926 1676 1695CTAGATTGTGACCATCTAAA 56 10648 10667 2337 563927 1677 1696TCTAGATTGTGACCATCTAA 37 10649 10668 2338 563928 1678 1697ATCTAGATTGTGACCATCTA 46 10650 10669 2339 563929 1679 1698AATCTAGATTGTGACCATCT 56 10651 10670 2340 563930 1680 1699TAATCTAGATTGTGACCATC 46 10652 10671 2341 563931 1681 1700ATAATCTAGATTGTGACCAT 35 10653 10672 2342 563932 1682 1701TATAATCTAGATTGTGACCA 45 10654 10673 2343 563933 1683 1702TTATAATCTAGATTGTGACC 37 10655 10674 2344 563934 1686 1705TGATTATAATCTAGATTGTG 28 10658 10677 2345 563935 1687 1706TTGATTATAATCTAGATTGT 0 10659 10678 2346 563936 1688 1707ATTGATTATAATCTAGATTG 0 10660 10679 2347 563937 1689 1708TATTGATTATAATCTAGATT 0 10661 10680 2348 563938 1690 1709CTATTGATTATAATCTAGAT 5 10662 10681 2349 563939 1691 1710CCTATTGATTATAATCTAGA 0 10663 10682 2350 563940 1692 1711ACCTATTGATTATAATCTAG 9 10664 10683 2351 563941 1693 1712CACCTATTGATTATAATCTA 5 10665 10684 2352 563942 1694 1713TCACCTATTGATTATAATCT 0 10666 10685 2353 563943 1695 1714TTCACCTATTGATTATAATC 10 10667 10686 2354 563944 1696 1715GTTCACCTATTGATTATAAT 31 10668 10687 2355 563945 1697 1716AGTTCACCTATTGATTATAA 15 10669 10688 2356 563946 1698 1717AAGTTCACCTATTGATTATA 31 10670 10689 2357 563947 1700 1719ATAAGTTCACCTATTGATTA 9 10672 10691 2358 563948 1701 1720AATAAGTTCACCTATTGATT 5 10673 10692 2359 563949 1702 1721TAATAAGTTCACCTATTGAT 14 10674 10693 2360 563950 1703 1722TTAATAAGTTCACCTATTGA 0 10675 10694 2361

TABLE 136 Inhibition of ANGPTL3 mRNA by 5-10-5 MOE gapmers targeting SEQID NO: 1 and 2 SEQ SEQ SEQ ID SEQ ID ID NO: NO: ID NO: NO: 2 SEQ ISIS 1Start 1 Stop % 2 Start Stop ID NO Site Site Sequence inhibition SiteSite NO 567064 N/A N/A TGAGTATTCTCGACTTACCT 26 8770 8789 2362 567065 N/AN/A AAGTGAGTATTCTCGACTTA 2 8773 8792 2363 567066 N/A N/AATTAAGTGAGTATTCTCGAC 20 8776 8795 2364 567067 N/A N/ACCAGAATTAAGTGAGTATTC 36 8781 8800 2365 567068 N/A N/AGCTTTCTTACCAGAATTAAG 75 8790 8809 84 567069 N/A N/A GTTGCTTTCTTACCAGAATT78 8793 8812 85 567070 N/A N/A TGGGTTGCTTTCTTACCAGA 26 8796 8815 2366567071 N/A N/A AAATGGGTTGCTTTCTTACC 3 8799 8818 2367 567072 N/A N/ATACAAATGGGTTGCTTTCTT 24 8802 8821 2368 567073 N/A N/AAAGTACAAATGGGTTGCTTT 24 8805 8824 2369 567074 N/A N/AGTAAATACAAGTACAAATGG 7 8813 8832 2370 567075 N/A N/ATTGCTGGTAAATACAAGTAC 24 8819 8838 2371 567076 N/A N/ATAAGGATTGCTGGTAAATAC 6 8825 8844 2372 567077 N/A N/ATTTTAAGGATTGCTGGTAAA 4 8828 8847 2373 567078 N/A N/AGCTTCATTTTAAGGATTGCT 60 8834 8853 87 567079 N/A N/A GAAGCTTCATTTTAAGGATT0 8837 8856 2374 567080 N/A N/A TAGGAAGCTTCATTTTAAGG 9 8840 8859 2375567081 N/A N/A TAGTAGGAAGCTTCATTTTA 18 8843 8862 2376 567082 N/A N/ATTGAGTTAGTAGGAAGCTTC 30 8849 8868 2377 567083 N/A N/AATTGCTATTGAGTTAGTAGG 21 8856 8875 2378 567084 N/A N/ACTTATTGCTATTGAGTTAGT 28 8859 8878 2379 567085 N/A N/AATTGTCTTATTGCTATTGAG 16 8864 8883 2380 567086 N/A N/AACTATTGTCTTATTGCTATT 10 8867 8886 2381 567087 N/A N/ATTCACTATTGTCTTATTGCT 35 8870 8889 2382 567088 N/A N/AACATTCACTATTGTCTTATT 30 8873 8892 2383 567089 N/A N/ATAAACATTCACTATTGTCTT 58 8876 8895 2384 567090 N/A N/ACATTAAACATTCACTATTGT 28 8879 8898 2385 567091 N/A N/AGTTTTCATTAAACATTCACT 54 8884 8903 2386 567092 N/A N/AAAATACTGTTTTCATTAAAC 34 8891 8910 2387 567093 N/A N/AAAAGTATTTATAAAATACTG 0 8903 8922 2388 567094 N/A N/ACCTTTTTATTAAAGTATTTA 0 8913 8932 2389 567095 N/A N/ACAATCCTTTTTATTAAAGTA 10 8917 8936 2390 567096 N/A N/ACTTCATCACAATCCTTTTTA 52 8925 8944 2391 567097 N/A N/AGTTCTTCATCACAATCCTTT 57 8928 8947 2392 567098 N/A N/AATTGTTCTTCATCACAATCC 37 8931 8950 2393 567099 N/A N/ATAGATTGTTCTTCATCACAA 31 8934 8953 2394 567100 N/A N/AAAATAGATTGTTCTTCATCA 11 8937 8956 2395 567101 N/A N/AAACAAATATAAATAGATTGT 0 8946 8965 2396 567102 N/A N/ACAAATAACAAATATAAATAG 3 8951 8970 2397 567103 N/A N/ATGGAATTAAAAACAAATAAC 3 8963 8982 2398 567104 N/A N/ATTATTGGAATTAAAAACAAA 12 8967 8986 2399 567105 N/A N/ATTTTTATTGGAATTAAAAAC 17 8970 8989 2400 567106 N/A N/ATAATAACTTTTTTCTGTAAT 6 9001 9020 2401 567107 N/A N/AGTTCTTAATAACTTTTTTCT 21 9006 9025 2402 567108 N/A N/AAAAAGCATGGTTCTTAATAA 0 9015 9034 2403 567109 N/A N/AAAATTTAAAAGCATGGTTCT 0 9021 9040 2404 567110 N/A N/AAGGAATAAATTTAAAAAATC 0 9046 9065 2405 567111 N/A N/AAGACAGGAATAAATTTAAAA 7 9050 9069 2406 567112 N/A N/AAAAAGACAGGAATAAATTTA 0 9053 9072 2407 567113 N/A N/ACTTTCTTTGTAGAAAAAGAC 29 9066 9085 2408 567114 N/A N/AATGCTTTCTTTGTAGAAAAA 12 9069 9088 2409 567115 N/A N/AGCTTAATGTATGCTTTCTTT 67 9078 9097 88 567116 N/A N/A TTTGCTTAATGTATGCTTTC21 9081 9100 2410 567117 N/A N/A GTATTTGCTTAATGTATGCT 0 9084 9103 2411567118 N/A N/A TTGGTATTTGCTTAATGTAT 0 9087 9106 2412 567119 N/A N/ACCTTTGGTATTTGCTTAATG 35 9090 9109 2413 567120 N/A N/ATGGCCTTTGGTATTTGCTTA 0 9093 9112 2414 567121 N/A N/ATAAACCTGGCCTTTGGTATT 27 9099 9118 2415 567122 N/A N/AATGTAAACCTGGCCTTTGGT 16 9102 9121 2416 567123 N/A N/ACAAATGTAAACCTGGCCTTT 0 9105 9124 2417 567124 N/A N/ACTTCAAATGTAAACCTGGCC 25 9108 9127 2418 567125 N/A N/ATTTCTTCAAATGTAAACCTG 2 9111 9130 2419 567126 N/A N/ATGTCACTTTCTTCAAATGTA 57 9117 9136 2420 567127 N/A N/ATAATGTCACTTTCTTCAAAT 6 9120 9139 2421 567128 N/A N/AAATAATAATGTCACTTTCTT 3 9125 9144 2422 567129 N/A N/AGAGTAATAATAATGTCACTT 18 9129 9148 2423 567130 N/A N/AGACTTGAGTAATAATAATGT 1 9134 9153 2424 567131 N/A N/ACCTAGAGACTTGAGTAATAA 32 9140 9159 2425 567132 N/A N/AATTCCTAGAGACTTGAGTAA 8 9143 9162 2426 567133 N/A N/AAAGTATTCCTAGAGACTTGA 11 9147 9166 2427 567134 N/A N/AGTTAAGTATTCCTAGAGACT 61 9150 9169 89 567135 N/A N/A TGTGTTAAGTATTCCTAGAG28 9153 9172 2428 567136 N/A N/A AGAGATGTGTTAAGTATTCC 31 9158 9177 2429567137 N/A N/A GTCAAGAGATGTGTTAAGTA 52 9162 9181 2430 567138 N/A N/AACAGTCAAGAGATGTGTTAA 22 9165 9184 2431 567139 N/A N/ATATACAGTCAAGAGATGTGT 30 9168 9187 2432 567140 N/A N/ACCATATACAGTCAAGAGATG 45 9171 9190 2433 567141 N/A N/AGTAAGTTGAACTAACTACTG 9 7497 7516 2434 567142 N/A N/ATGAGTAAGTTGAACTAACTA 0 7500 7519 2435 567143 N/A N/ATAATGAGTAAGTTGAACTAA 2 7503 7522 2436 567144 N/A N/AAGGTTAATCTTCCTAATACG 18 7523 7542 2437 567145 N/A N/AATAACCAGGTTAATCTTCCT 34 7529 7548 2438 567146 N/A N/AATGATAACCAGGTTAATCTT 13 7532 7551 2439 567147 N/A N/AAACAATGATAACCAGGTTAA 7 7536 7555 2440 567148 N/A N/ATAAAACAATGATAACCAGGT 45 7539 7558 2441 567149 N/A N/AGTATAAAACAATGATAACCA 26 7542 7561 2442 567150 N/A N/ACGAATACTCATATATATTTC 25 7572 7591 2443 567151 N/A N/AATACGAATACTCATATATAT 30 7575 7594 2444 567152 N/A N/ATTTATACGAATACTCATATA 32 7578 7597 2445 567153 N/A N/AATATTTATACGAATACTCAT 25 7581 7600 2446 567154 N/A N/AGTATTATATTTATACGAATA 0 7586 7605 2447 567155 N/A N/AAAAAGTATTATATTTATACG 0 7590 7609 2448 567156 N/A N/AGGTAAAAGTATTATATTTAT 0 7593 7612 2449 567157 N/A N/AACAAGGTAAAAGTATTATAT 10 7597 7616 2450 567158 N/A N/ATAAACAAGGTAAAAGTATTA 11 7600 7619 2451 567159 N/A N/AACATAAACAAGGTAAAAGTA 3 7603 7622 2452 567160 N/A N/ATTGAGTAAATACATAAACAA 12 7613 7632 2453 567161 N/A N/AGAGAATATTGAGTAAATACA 4 7620 7639 2454 567162 N/A N/AAAGGAGAATATTGAGTAAAT 8 7623 7642 2455 567163 N/A N/AGAAAAGGAGAATATTGAGTA 3 7626 7645 2456 567164 N/A N/AGAGGAAAAGGAGAATATTGA 19 7629 7648 2457 567165 N/A N/ATTAGAGGAAAAGGAGAATAT 41 7632 7651 2458 567166 N/A N/AATTATTTTAGAGGAAAAGGA 30 7638 7657 2459 567167 N/A N/ACAGATTATTTTAGAGGAAAA 9 7641 7660 2460 567168 N/A N/ACTTCAGATTATTTTAGAGGA 24 7644 7663 2461 567169 N/A N/ATAGTCACTTCAGATTATTTT 38 7650 7669 2462 567170 N/A N/ATAATAGTCACTTCAGATTAT 13 7653 7672 2463 567171 N/A N/ATGATAATAGTCACTTCAGAT 39 7656 7675 2464 567172 N/A N/ATATTGATAATAGTCACTTCA 41 7659 7678 2465 567173 N/A N/AACTTATTGATAATAGTCACT 29 7662 7681 2466 567174 N/A N/ATAAACTTATTGATAATAGTC 14 7665 7684 2467 567175 N/A N/ATAGTAAACTTATTGATAATA 31 7668 7687 2468 567176 N/A N/AGCATAGTAAACTTATTGATA 23 7671 7690 2469 567177 N/A N/ATTGGCATAGTAAACTTATTG 21 7674 7693 2470 567178 N/A N/AATTTTGGCATAGTAAACTTA 8 7677 7696 2471 567179 N/A N/ATGAATTTTGGCATAGTAAAC 5 7680 7699 2472 567180 N/A N/ATTAATGAATTTTGGCATAGT 0 7684 7703 2473 567181 N/A N/ACAATTAATGAATTTTGGCAT 39 7687 7706 2474 567182 N/A N/AAAAGGCAATTAATGAATTTT 12 7692 7711 2475 567183 N/A N/AGTGAAAGGCAATTAATGAAT 28 7695 7714 2476 567184 N/A N/ATTAAGTGAAAGGCAATTAAT 7 7699 7718 2477 567185 N/A N/AAAGTTAAGTGAAAGGCAATT 25 7702 7721 2478 567186 N/A N/ACCAAAAGTTAAGTGAAAGGC 50 7706 7725 2479 567187 N/A N/AGTCCCAAAAGTTAAGTGAAA 30 7709 7728 2480 567188 N/A N/AATGGTCCCAAAAGTTAAGTG 39 7712 7731 2481 567189 N/A N/AATTATGGTCCCAAAAGTTAA 19 7715 7734 2482 567190 N/A N/ATTTATTATGGTCCCAAAAGT 33 7718 7737 2483 567191 N/A N/ATTATTATTTATTATGGTCCC 50 7724 7743 2484 567192 N/A N/AATGGCAATACATTTTATTAT 13 7737 7756 2485 567193 N/A N/AGTTATGGCAATACATTTTAT 39 7740 7759 2486 567194 N/A N/ATAATGTTATGGCAATACATT 0 7744 7763 2487 567195 N/A N/ATATTAATGTTATGGCAATAC 22 7747 7766 2488 567196 N/A N/AGTTTATTAATGTTATGGCAA 28 7750 7769 2489 567197 N/A N/AGTAGTTTATTAATGTTATGG 20 7753 7772 2490 567198 N/A N/AAAGGTAGTTTATTAATGTTA 27 7756 7775 2491 567199 N/A N/ATGTAAGGTAGTTTATTAATG 0 7759 7778 2492 567200 N/A N/ATTTTGTAAGGTAGTTTATTA 0 7762 7781 2493 567201 N/A N/ATGGTTTTGTAAGGTAGTTTA 18 7765 7784 2494 567202 N/A N/ATGGTGGTTTTGTAAGGTAGT 0 7768 7787 2495 567203 N/A N/AAATTGGTGGTTTTGTAAGGT 11 7771 7790 2496 567204 N/A N/ATTTAATTGGTGGTTTTGTAA 0 7774 7793 2497 567205 N/A N/ATTGATTTTAATTGGTGGTTT 19 7779 7798 2498 567206 N/A N/ATGTTTGATTTTAATTGGTGG 26 7782 7801 2499 567207 N/A N/AATGTAAATAACACTTTTTTG 1 7804 7823 2500 567208 N/A N/ACAGATGTAAATAACACTTTT 1 7807 7826 2501 567209 N/A N/ATGACAGATGTAAATAACACT 21 7810 7829 2502 567210 N/A N/AATGTTGACAGATGTAAATAA 0 7814 7833 2503 567211 N/A N/ATTTATGTTGACAGATGTAAA 0 7817 7836 2504 567212 N/A N/AAGATTTATGTTGACAGATGT 0 7820 7839 2505 567213 N/A N/AAGTAGATTTATGTTGACAGA 19 7823 7842 2506 567214 N/A N/ATTTAGTAGATTTATGTTGAC 4 7826 7845 2507 567215 N/A N/AATTTTTAGTAGATTTATGTT 0 7829 7848 2508 567216 N/A N/ACATGTATTTTTAGTAGATTT 5 7834 7853 2509 567217 N/A N/AGAAATCATGTATTTTTAGTA 0 7839 7858 2510 567218 N/A N/AATTGTATTTGATGGATATCT 43 6875 6894 2511 567219 N/A N/AGATACATTGTATTTGATGGA 20 6880 6899 2512 567220 N/A N/ATAGGTTGATACATTGTATTT 18 6886 6905 2513 567221 N/A N/ACAGTTTAGGTTGATACATTG 18 6891 6910 2514 567222 N/A N/AGCATCCAGTTTAGGTTGATA 31 6896 6915 2515 567223 N/A N/ACCCCAGCATCCAGTTTAGGT 14 6901 6920 2516 567224 N/A N/AAAGAACCCCAGCATCCAGTT 41 6906 6925 2517 567225 N/A N/AGTGTAAAAAGAACCCCAGCA 0 6913 6932 2518 567226 N/A N/AATAGGGTGTAAAAAGAACCC 13 6918 6937 2519 567227 N/A N/ACTTTTATAGGGTGTAAAAAG 0 6923 6942 2520 567228 N/A N/ATATGTCTTTTATAGGGTGTA 26 6928 6947 2521 567229 N/A N/ATTAGGTATGTCTTTTATAGG 0 6933 6952 2522 567230 N/A N/ATTGTCTTAGGTATGTCTTTT 30 6938 6957 2523 567231 N/A N/ACTCTGATTGTCTTAGGTATG 27 6944 6963 2524 567232 N/A N/ATATTTCTCTGATTGTCTTAG 21 6949 6968 2525 567233 N/A N/ATCCATATTTGTATTTCTCTG 61 6959 6978 90 567234 N/A N/A TCAAGTCCATATTTGTATTT20 6964 6983 2526 567235 N/A N/A AATAATCAAGTCCATATTTG 0 6969 6988 2527567236 N/A N/A TTATCTAATAATCAAGTCCA 0 6975 6994 2528 567237 N/A N/ACTATATTATCTAATAATCAA 12 6980 6999 2529 567238 N/A N/ATAAACCTTCTATATTATCTA 12 6988 7007 2530 567239 N/A N/AAATTAATAAACCTTCTATAT 0 6994 7013 2531 567240 N/A N/ATAAGTACAGGTTGGACACTG 0 9504 9523 2532 567241 N/A N/AGTTATTAAGTACAGGTTGGA 2 9509 9528 2533 567242 N/A N/ATGTGAGTTATTAAGTACAGG 0 9514 9533 2534 567243 N/A N/AAAATCTGTGAGTTATTAAGT 0 9519 9538 2535 567244 N/A N/AGTTTTAAAAATCTGTGAGTT 19 9526 9545 2536 567245 N/A N/ACAAAATTCTCCTGAAAAGAA 20 9548 9567 2537 567246 N/A N/ACCCAACCAAAATTCTCCTGA 48 9554 9573 2538 567247 N/A N/AACCTGAATAACCCTCTGGAC 21 9807 9826 2539 567248 N/A N/AAAGATACCTGAATAACCCTC 30 9812 9831 2540 567249 N/A N/AAGAAAAAGATACCTGAATAA 0 9817 9836 2541 567250 N/A N/ATGGTATCAGAAAAAGATACC 0 9824 9843 2542 567251 N/A N/AAGTATTGGTATCAGAAAAAG 0 9829 9848 2543 567252 N/A N/AAATAAAGTATTGGTATCAGA 10 9834 9853 2544 567253 N/A N/AATGAAAATAAAGTATTGGTA 3 9839 9858 2545 567254 N/A N/AAGATACTTTGAAGATATGAA 0 9854 9873 2546 567255 N/A N/ATGGGAAGATACTTTGAAGAT 0 9859 9878 2547 567256 N/A N/ACTAATAATGTGGGAAGATAC 0 9868 9887 2548 567257 N/A N/ACATTGCAGATAATAGCTAAT 0 9883 9902 2549 567258 N/A N/AAAGTTGTCATTGCAGATAAT 0 9890 9909 2550 567259 N/A N/ATTTTAAAAGTTGTCATTGCA 7 9896 9915 2551 567260 N/A N/AATTCGGATTTTTAAAAGTTG 5 9904 9923 2552 567261 N/A N/ATTATTTGGGATTCGGATTTT 15 9913 9932 2553 567262 N/A N/ATTATAGTTAAGAGGTTTTCG 27 9949 9968 2554 567263 N/A N/ATTTCATTATAGTTAAGAGGT 12 9954 9973 2555 567264 N/A N/AGAACACTTTCATTATAGTTA 13 9960 9979 2556 567265 N/A N/AGAACTAGAATGAACACTTTC 28 9970 9989 2557 567266 N/A N/ATGATTGAACTAGAATGAACA 23 9975 9994 2558 567267 N/A N/AATACCTGATTGAACTAGAAT 9 9980 9999 2559 567268 N/A N/AGTAAAATACCTGATTGAACT 6 9985 10004 2560 567269 N/A N/ATAGAGGTAAAATACCTGATT 16 9990 10009 2561 567270 N/A N/AAAGATTAGAGGTAAAATACC 0 9995 10014 2562 567271 N/A N/ATGAGGAAGATTAGAGGTAAA 6 10000 10019 2563 567272 N/A N/AGAAAATCTGAGGAAGATTAG 0 10007 10026 2564 567273 N/A N/AAAATAGAAAATCTGAGGAAG 0 10012 10031 2565 567274 N/A N/AATCTATACACTACCAAAAAA 0 10029 10048 2566 567275 N/A N/AAAATAATCTATACACTACCA 19 10034 10053 2567 567276 N/A N/AAAATAATCTGTATAAATAAT 3 10047 10066 2568 567277 N/A N/ACCCAATTTTAAATAATCTGT 24 10056 10075 2569 567278 N/A N/ATAAGTCCCAATTTTAAATAA 0 10061 10080 2570 567279 N/A N/ATCTGTATAAGTCCCAATTTT 15 10067 10086 2571 567280 N/A N/AAATAATCTGTATAAGTCCCA 47 10072 10091 2572 567281 N/A N/AAGTTTTAAATAATCTGTATA 0 10079 10098 2573 567282 N/A N/AATCCCAGTTTTAAATAATCT 6 10084 10103 2574 567283 N/A N/ACATGTATCCCAGTTTTAAAT 6 10089 10108 2575 567284 N/A N/ATAGATGCATGTATCCCAGTT 41 10095 10114 2576 567285 N/A N/ATGTTTTAGATGCATGTATCC 4 10100 10119 2577 567286 N/A N/ATACAGTGTTTTAGATGCATG 25 10105 10124 2578 567287 N/A N/AAATATTACAGTGTTTTAGAT 0 10110 10129 2579 567288 N/A N/ACTTATAAATATTACAGTGTT 2 10116 10135 2580 567289 N/A N/ACTTCCTTTCTTATAAATATT 12 10124 10143 2581 567290 N/A N/ATTTATCTTCCTTTCTTATAA 0 10129 10148 2582 567291 N/A N/ACGTAAGTTTATCTTCCTTTC 61 10135 10154 91 567292 N/A N/ATTCCCCGTAAGTTTATCTTC 22 10140 10159 2583 567293 N/A N/ATGTATTTCCCCGTAAGTTTA 0 10145 10164 2584 567294 N/A N/AGTTACTGTATTTCCCCGTAA 43 10150 10169 2585 544120 707 726AGTTCTTGGTGCTCTTGGCT 80 6720 6739 15 337487 804 823 CACTTGTATGTTCACCTCTG80 7389 7408 28

TABLE 137 Inhibition of ANGPTL3 mRNA by 5-10-5 MOE gapmers targeting SEQID NO: 1 and 2 SEQ SEQ SEQ ID ID SEQ ID ID NO: 1 NO: NO: 2 NO: 2 SEQISIS Start 1 Stop % Start Stop ID NO Site Site Sequence inhibition SiteSite NO 563780 N/A N/A TGTTTTCTTCTGGAAGCAGA 10 3100 3119 2586 568085 N/AN/A CAGACCTAGACTTCTTAACT 8 3084 3103 2587 568086 N/A N/AAGCAGACCTAGACTTCTTAA 6 3086 3105 2588 568087 N/A N/ATTTTCTTCTGGAAGCAGACC 0 3098 3117 2589 568088 N/A N/AAAACATATATACATGCTTGT 52 11323 11342 2590 568089 N/A N/ATTAAACATATATACATGCTT 39 11325 11344 2591 568090 N/A N/AGTTTATTGAATTTTAAACAT 0 11337 11356 2592 568091 N/A N/ATTGTTTATTGAATTTTAAAC 9 11339 11358 2593 568092 N/A N/ACTTTGTTTATTGAATTTTAA 0 11341 11360 2594 568093 N/A N/AGTCTTTGTTTATTGAATTTT 28 11343 11362 2595 568094 N/A N/AGGGTCTTTGTTTATTGAATT 0 11345 11364 2596 568095 N/A N/ACTGGGTCTTTGTTTATTGAA 11 11347 11366 2597 568096 N/A N/AGACTGGGTCTTTGTTTATTG 35 11349 11368 2598 568097 N/A N/ATTTCTATAATTTAGGGACTG 12 11364 11383 2599 568098 N/A N/AAATTTCTATAATTTAGGGAC 0 11366 11385 2600 568099 N/A N/ATAAATTTCTATAATTTAGGG 5 11368 11387 2601 568100 N/A N/ACAAGAATAATTTAAATTTCT 38 11379 11398 2602 568101 N/A N/AGATAAACATGCAAGAATAAT 1 11389 11408 2603 568102 N/A N/ATCGATAAACATGCAAGAATA 51 11391 11410 2604 568103 N/A N/ATGTCGATAAACATGCAAGAA 37 11393 11412 2605 568104 N/A N/AGATGTCGATAAACATGCAAG 57 11395 11414 2606 568105 N/A N/AGTGATGTCGATAAACATGCA 61 11397 11416 2607 568106 N/A N/ATTGTGATGTCGATAAACATG 57 11399 11418 2608 568107 N/A N/ATGTTGTGATGTCGATAAACA 47 11401 11420 2609 568108 N/A N/ATCTGTTGTGATGTCGATAAA 53 11403 11422 2610 568109 N/A N/AGATCTGTTGTGATGTCGATA 36 11405 11424 2611 568110 N/A N/AGGGATCTGTTGTGATGTCGA 41 11407 11426 2612 568111 N/A N/ATAGGGATCTGTTGTGATGTC 43 11409 11428 2613 568112 N/A N/ATTTAGGGATCTGTTGTGATG 18 11411 11430 2614 568113 N/A N/AGATTTAGGGATCTGTTGTGA 41 11413 11432 2615 568114 N/A N/AATCTAATCTTTAGGGATTTA 37 11435 11454 2616 568115 N/A N/ATTTGTATCTAATCTTTAGGG 28 11440 11459 2617 568116 N/A N/AAATTTGTATCTAATCTTTAG 0 11442 11461 2618 568117 N/A N/AGTGGTAAAAAATTTGTATCT 13 11451 11470 2619 568118 N/A N/ACTGTGGTAAAAAATTTGTAT 5 11453 11472 2620 568119 N/A N/ATACTGTGGTAAAAAATTTGT 10 11455 11474 2621 568120 N/A N/AGATACTGTGGTAAAAAATTT 17 11457 11476 2622 568121 N/A N/AAGTGATACTGTGGTAAAAAA 38 11460 11479 2623 568122 N/A N/ACAAGTGATACTGTGGTAAAA 58 11462 11481 2624 568123 N/A N/AGACAAGTGATACTGTGGTAA 52 11464 11483 2625 568124 N/A N/ACTGACAAGTGATACTGTGGT 62 11466 11485 2626 568125 N/A N/ATTCTGACAAGTGATACTGTG 27 11468 11487 2627 568126 N/A N/AAATTCTGACAAGTGATACTG 33 11470 11489 2628 568127 N/A N/AATAAATTCTGACAAGTGATA 38 11473 11492 2629 568128 N/A N/ACTGGCAGTTTTAAAAAATCA 28 11502 11521 2630 568129 N/A N/ATTCTTACTGGCAGTTTTAAA 56 11508 11527 2631 568130 N/A N/AATTTCTTACTGGCAGTTTTA 47 11510 11529 2632 568131 N/A N/AAAATTTCTTACTGGCAGTTT 53 11512 11531 2633 568132 N/A N/ATTTAAAATTTCTTACTGGCA 46 11516 11535 2634 568133 N/A N/ATTAATTTAAAATTTCTTACT 9 11520 11539 2635 568134 N/A N/ACAAATGGGTTTAATTTAAAA 1 11529 11548 2636 568135 N/A N/AAACAAATGGGTTTAATTTAA 11 11531 11550 2637 568136 N/A N/ATTAACAAATGGGTTTAATTT 12 11533 11552 2638 568137 N/A N/ACTTTAACAAATGGGTTTAAT 27 11535 11554 2639 568138 N/A N/ATCCTTTAACAAATGGGTTTA 52 11537 11556 2640 568139 N/A N/ACTATATCCTTTAACAAATGG 24 11542 11561 2641 568140 N/A N/AGGGCACTATATCCTTTAACA 45 11547 11566 2642 568141 N/A N/ATTGGGCACTATATCCTTTAA 20 11549 11568 2643 568142 N/A N/ATATAACTTGGGCACTATATC 27 11555 11574 2644 568143 N/A N/ACATATAACTTGGGCACTATA 40 11557 11576 2645 568144 N/A N/AACCATATAACTTGGGCACTA 69 11559 11578 103 568145 N/A N/ATCACCATATAACTTGGGCAC 60 11561 11580 2646 568146 N/A N/AGGTCACCATATAACTTGGGC 73 11563 11582 104 568147 N/A N/ATAGGTCACCATATAACTTGG 51 11565 11584 2647 568148 N/A N/AGGTAGGTCACCATATAACTT 57 11567 11586 2648 568149 N/A N/AAAGGTAGGTCACCATATAAC 52 11569 11588 2649 568150 N/A N/ACAAAGGTAGGTCACCATATA 28 11571 11590 2650 568151 N/A N/AGACAAAGGTAGGTCACCATA 67 11573 11592 105 568152 N/A N/AGTATTGACAAAGGTAGGTCA 55 11578 11597 2651 568153 N/A N/AAAGTATTGACAAAGGTAGGT 36 11580 11599 2652 568154 N/A N/ACTAAGTATTGACAAAGGTAG 24 11582 11601 2653 568155 N/A N/ATGCTAAGTATTGACAAAGGT 49 11584 11603 2654 568156 N/A N/AAATGCTAAGTATTGACAAAG 10 11586 11605 2655 568157 N/A N/ACATAATGCTAAGTATTGACA 19 11589 11608 2656 568158 N/A N/ATACATAATGCTAAGTATTGA 4 11591 11610 2657 568159 N/A N/AAATACATAATGCTAAGTATT 1 11593 11612 2658 568160 N/A N/AGAAATACATAATGCTAAGTA 23 11595 11614 2659 568161 N/A N/ATTTGAAATACATAATGCTAA 8 11598 11617 2660 568162 N/A N/AGGATAATTTGAAATACATAA 16 11604 11623 2661 568163 N/A N/ATTGGATAATTTGAAATACAT 0 11606 11625 2662 568164 N/A N/ATATTGGATAATTTGAAATAC 0 11608 11627 2663 568165 N/A N/AATCCAGTTAAAGCTTGTAAA 46 4466 4485 2664 568166 N/A N/ATCATGATCCAGTTAAAGCTT 32 4471 4490 2665 568167 N/A N/ATTTACTCATGATCCAGTTAA 24 4476 4495 2666 568168 N/A N/AGATAATTTTACTCATGATCC 53 4482 4501 2667 568169 N/A N/AGATGTGATAATTTTACTCAT 27 4487 4506 2668 568170 N/A N/AATGCTGATGTGATAATTTTA 42 4492 4511 2669 568171 N/A N/ACAGTTATGCTGATGTGATAA 0 4497 4516 2670 568172 N/A N/ATTTAACAGTTATGCTGATGT 17 4502 4521 2671 568173 N/A N/AGCAATTTTAACAGTTATGCT 11 4507 4526 2672 568174 N/A N/AAGAGCCTGCAATTTTAACAG 25 4514 4533 2673 568175 N/A N/AGCTTCAGAGCCTGCAATTTT 47 4519 4538 2674 568176 N/A N/ATATTAGCTTCAGAGCCTGCA 48 4524 4543 2675 568177 N/A N/ATAGTTTATTAGCTTCAGAGC 20 4529 4548 2676 568178 N/A N/AGCAGGTAGTTTATTAGCTTC 39 4534 4553 2677 568179 N/A N/ATAAATGCAGGTAGTTTATTA 0 4539 4558 2678 568180 N/A N/AATGGTTTAAATGCAGGTAGT 20 4545 4564 2679 568181 N/A N/AGAGCCATGGTTTAAATGCAG 33 4550 4569 2680 568182 N/A N/ATTTTAGAGCCATGGTTTAAA 40 4555 4574 2681 568183 N/A N/ACAAAGTTTTAGAGCCATGGT 54 4560 4579 2682 568184 N/A N/ATCACACAAAGTTTTAGAGCC 61 4565 4584 2683 568185 N/A N/ACAAGGTCACACAAAGTTTTA 17 4570 4589 2684 568186 N/A N/AGGGTGAAGTAATTTATTCAA 0 4587 4606 2685 568187 N/A N/AGTGAGGAAACTGAGAGATAA 12 4609 4628 2686 568188 N/A N/ATGTAGTATATGTGAGGAAAC 38 4619 4638 2687 568189 N/A N/AATCTTTGTAGTATATGTGAG 30 4624 4643 2688 568190 N/A N/ATTATTATCTTTGTAGTATAT 19 4629 4648 2689 568191 N/A N/ATTCTGTTATTATCTTTGTAG 48 4634 4653 2690 568192 N/A N/AATAAGTTCTGTTATTATCTT 16 4639 4658 2691 568193 N/A N/AATCCTATAAGTTCTGTTATT 22 4644 4663 2692 568194 N/A N/ACAATAATCCTATAAGTTCTG 0 4649 4668 2693 568195 N/A N/ATAAGATGACATTGGCTGCTA 49 4689 4708 2694 568196 N/A N/ATTTAGTAAGATGACATTGGC 32 4694 4713 2695 568197 N/A N/ATTGAATTTTAGTAAGATGAC 19 4700 4719 2696 568198 N/A N/ACTAATTTGAATTTTAGTAAG 34 4705 4724 2697 568199 N/A N/ACATGATCTAATTTGAATTTT 29 4711 4730 2698 568200 N/A N/ACAAAGAGAAACATGATCTAA 27 4721 4740 2699 568201 N/A N/AGTTTTGAGCAAAGAGAAACA 36 4729 4748 2700 568202 N/A N/AGTGTGGTTTTGAGCAAAGAG 3 4734 4753 2701 568203 N/A N/AAGCTATTGTGTGGTTTTGAG 13 4741 4760 2702 568204 N/A N/ATGAAATGGAAAGCTATTGTG 15 4751 4770 2703 568205 N/A N/ATATGAGTGAAATGGAAAGCT 27 4757 4776 2704 568206 N/A N/AGCCAATATGAGTGAAATGGA 62 4762 4781 106 568207 N/A N/AAAAGAGCCAATATGAGTGAA 25 4767 4786 2705 568208 N/A N/ATTGGTCTAAAGAGCCAATAT 42 4774 4793 2706 568209 N/A N/AGGTAATCTTGGTCTAAAGAG 29 4781 4800 2707 568210 N/A N/AGTGAGATGACGAAGGGTTGG 0 4800 4819 2708 568211 N/A N/AAGTCAGTGAGATGACGAAGG 5 4805 4824 2709 568212 N/A N/AGGTGAAGTCAGTGAGATGAC 12 4810 4829 2710 568213 N/A N/AGTAGAGGAGGTGAAGTCAGT 13 4818 4837 2711 568214 N/A N/AAACTAGAGTAGAGGAGGTGA 20 4825 4844 2712 568215 N/A N/AAGAATAACTAGAGTAGAGGA 33 4830 4849 2713 568216 N/A N/ACGGTCAGAATAACTAGAGTA 39 4835 4854 2714 568217 N/A N/ATAAAGCGGTCAGAATAACTA 29 4840 4859 2715 568218 N/A N/AACTGGTAAAGCGGTCAGAAT 17 4845 4864 2716 568219 N/A N/ATGAATACTGGTAAAGCGGTC 37 4850 4869 2717 568220 N/A N/ATGTGTTTGAATACTGGTAAA 21 4856 4875 2718 568221 N/A N/AAGTATGTTTGATGTGTTTGA 25 4867 4886 2719 568222 N/A N/AGTGGCAGTATGTTTGATGTG 15 4872 4891 2720 568223 N/A N/ATTGAGGTGGCAGTATGTTTG 14 4877 4896 2721 568224 N/A N/AAGGCTTTGAGGTGGCAGTAT 33 4882 4901 2722 568225 N/A N/AGGCAAAGGCTTTGAGGTGGC 27 4887 4906 2723 568226 N/A N/AAACAAGGGCAAAGGCTTTGA 24 4893 4912 2724 568227 N/A N/ATAGAGGAAACAACAAGGGCA 24 4903 4922 2725 568228 N/A N/ACCAGTTAGAGGAAACAACAA 4 4908 4927 2726 568229 N/A N/AGATACCAGGGCAGAAGAGCG 24 4930 4949 2727 568230 N/A N/AAAATCAGAGAGTGGGCCACG 24 4952 4971 2728 568231 N/A N/ACCTAAGGGAAATCAGAGAGT 19 4960 4979 2729 568232 N/A N/AACGACCCTAAGGGAAATCAG 30 4965 4984 2730 568233 N/A N/ATGATAACGACCCTAAGGGAA 0 4970 4989 2731 568234 N/A N/ATTTTGTTTGATAACGACCCT 22 4977 4996 2732 568235 N/A N/AGTCTTCATTGGGAATTTTTT 37 4993 5012 2733 568236 N/A N/ATGTAAGTCTTCATTGGGAAT 23 4998 5017 2734 568237 N/A N/AGACCTTGTAAGTCTTCATTG 52 5003 5022 2735 568238 N/A N/ATAAGTGACCTTGTAAGTCTT 36 5008 5027 2736 568239 N/A N/ATTGGTTAAGTGACCTTGTAA 11 5013 5032 2737 568240 N/A N/ATGATTTTTGGTTAAGTGACC 12 5019 5038 2738 568241 N/A N/AGGTTGTGATTTTTGGTTAAG 11 5024 5043 2739 568242 N/A N/ACAGGCGGTTGTGATTTTTGG 41 5029 5048 2740 568243 N/A N/AGGGACCAGGCGGTTGTGATT 22 5034 5053 2741 568244 N/A N/ACTAAGGAAGTAGAAGTTTTC 42 5060 5079 2742 568245 N/A N/AAGTAGCTAAGGAAGTAGAAG 11 5065 5084 2743 568246 N/A N/ACAGGAGAAAAGTAGCTAAGG 36 5074 5093 2744 568247 N/A N/AGTGTGCAGGAGAAAAGTAGC 14 5079 5098 2745 568248 N/A N/ATAAAGGTGAGTGTGCAGGAG 7 5088 5107 2746 568249 N/A N/AATGTTAAATAAAGGTGAGTG 8 5096 5115 2747 568250 N/A N/AATGTTATGTTAAATAAAGGT 27 5101 5120 2748 568251 N/A N/AAATTTATGTTATGTTAAATA 27 5106 5125 2749 568252 N/A N/ATAACTAAAATTTATGTTATG 28 5113 5132 2750 568253 N/A N/AGATAAATAACTAAAATTTAT 32 5119 5138 2751 568254 N/A N/ATTTAGTGCAGGAATAGAAGA 33 5139 5158 2752 568255 N/A N/AAATCCCTGTATTCACAGAGC 68 5165 5184 2753 568256 N/A N/AGAAAAAATCCCTGTATTCAC 0 5170 5189 2754 568257 N/A N/ATAATGGAAAAAATCCCTGTA 8 5175 5194 2755 568258 N/A N/AAAATATGAAGATAATGGAAA 26 5186 5205 2756 568259 N/A N/AATAATGGAAAATATGAAGAT 18 5194 5213 2757 568260 N/A N/ATATACAAATAATGGAAAATA 30 5201 5220 2758 568261 N/A N/ATTCTGGAGTATATACAAATA 45 5211 5230 2759 568262 N/A N/AATTCTATATTCTGGAGTATA 40 5219 5238 2760 568263 N/A N/ACCATACAGTATTCTATATTC 57 5228 5247 2761 568264 N/A N/ACTGTGTGCCATACAGTATTC 28 5235 5254 2762 568265 N/A N/AGCCTACTGTGTGCCATACAG 60 5240 5259 2763 568266 N/A N/AAGAAATGCCTACTGTGTGCC 42 5246 5265 2764 568267 N/A N/ATCAACAGAAATGCCTACTGT 52 5251 5270 2765 568268 N/A N/AATTAATTCAACAGAAATGCC 46 5257 5276 2766 568269 N/A N/AGACATTACATTTATTAATTC 32 5269 5288 2767 568270 N/A N/AGTGAATATGACATTACATTT 32 5277 5296 2768 568271 N/A N/ACTTCTGTGTGAATATGACAT 50 5284 5303 2769 568272 N/A N/AACACGCTTCTGTGTGAATAT 43 5289 5308 2770 568273 N/A N/AATAGCACACGCTTCTGTGTG 31 5294 5313 2771 568274 N/A N/ATAATCATAGCACACGCTTCT 40 5299 5318 2772 568275 N/A N/AAATAATAATCATAGCACACG 20 5304 5323 2773 568276 N/A N/ACCAAGTAATAATAATCATAG 35 5310 5329 2774 568277 N/A N/ACTAGTAATCCAAGTAATAAT 38 5318 5337 2775 568278 N/A N/ATATTTCTAGTAATCCAAGTA 39 5323 5342 2776 568279 N/A N/ACACACTATTTCTAGTAATCC 51 5328 5347 2777 568280 N/A N/ATTATGAGGCACACTATTTCT 25 5336 5355 2778 568281 N/A N/ATTTAATTATGAGGCACACTA 35 5341 5360 2779 568282 N/A N/AGTTGACCTTTAATTATGAGG 63 5348 5367 2780 568283 N/A N/ATTACATTGTTGAATGTTGAC 45 5362 5381 2781 568284 N/A N/AATTAATTACATTGTTGAATG 31 5367 5386 2782 568285 N/A N/ATGTAGATTAATTACATTGTT 49 5372 5391 2783 568286 N/A N/ATACATTGTAGATTAATTACA 43 5377 5396 2784 568287 N/A N/AAGATGTTTACATTGTAGATT 28 5384 5403 2785 568288 N/A N/ATTCACCAGATGTTTACATTG 36 5390 5409 2786 568289 N/A N/AGTCACTTCACCAGATGTTTA 65 5395 5414 2787 568290 N/A N/ACCTCTGTCACTTCACCAGAT 67 5400 5419 2788 568291 N/A N/AGCTTCCCTCTGTCACTTCAC 70 5405 5424 2789 568292 N/A N/ACAAGTGCTTCCCTCTGTCAC 33 5410 5429 2790 568293 N/A N/ATTTCTAAACAAGTGCTTCCC 70 5418 5437 107 568294 N/A N/AGCTTTTTTCTAAACAAGTGC 45 5423 5442 2791 568295 N/A N/AACATAGCTTTTTTCTAAACA 9 5428 5447 2792 568296 N/A N/ATTCTGACATAGCTTTTTTCT 23 5433 5452 2793 568297 N/A N/AATGGATTCTGACATAGCTTT 46 5438 5457 2794 568298 N/A N/AAATACATGGATTCTGACATA 37 5443 5462 2795 568299 N/A N/AATTAGAATACATGGATTCTG 57 5448 5467 2796 568300 N/A N/ACTGCATATTAGAATACATGG 75 5454 5473 108 568301 N/A N/ATTGTACTGCATATTAGAATA 53 5459 5478 2797 568302 N/A N/AAACTATTGTACTGCATATTA 25 5464 5483 2798 568303 N/A N/ATTTTAAACTATTGTACTGCA 25 5469 5488 2799 568304 N/A N/ATGAGAGTATTATTAATATTT 8 5487 5506 2800 568305 N/A N/AGCTGTTTGAGAGTATTATTA 50 5493 5512 2801 568306 N/A N/AGAATAGCTGTTTGAGAGTAT 38 5498 5517 2802 568307 N/A N/ACCTCTTGAATAGCTGTTTGA 55 5504 5523 2803 568308 N/A N/ATGAATCCTCTTGAATAGCTG 55 5509 5528 2804 568309 N/A N/ATTTTTTGAATCCTCTTGAAT 46 5514 5533 2805 568310 N/A N/ATTATGTTTTTTGAATCCTCT 36 5519 5538 2806 568311 N/A N/AGTTTATATTATGTTTTTTGA 6 5526 5545 2807 568312 N/A N/ATCTGAGTTTATATTATGTTT 29 5531 5550 2808 568313 N/A N/ACAGTTTCTCTGAGTTTATAT 28 5538 5557 2809 568314 N/A N/AGTTTACCAGTTTCTCTGAGT 44 5544 5563 2810 568315 N/A N/AATTTTGTTTACCAGTTTCTC 58 5549 5568 2811 568316 N/A N/AAAATGATTTTGTTTACCAGT 29 5554 5573 2812 568317 N/A N/ACTCTTGAAAATGATTTTGTT 22 5561 5580 2813 568318 N/A N/ATATATCTCTTGAAAATGATT 5 5566 5585 2814 568319 N/A N/ACAGGTTGGCAAGTTTGTTTG 27 6175 6194 2815 568320 N/A N/AGTTGGCAGGTTGGCAAGTTT 44 6180 6199 2816 568321 N/A N/AATATCTGTAGATGTTGGCAG 59 6192 6211 2817 568322 N/A N/ATAAACATATCTGTAGATGTT 18 6197 6216 2818 568323 N/A N/AACCTGTAAACATATCTGTAG 57 6202 6221 2819 568324 N/A N/ATTTTGACCTGTAAACATATC 23 6207 6226 2820 568325 N/A N/AATAATTTTTGACCTGTAAAC 7 6212 6231 2821 568326 N/A N/ATAATTTGATAATTTTTGACC 7 6219 6238 2822 568327 N/A N/ATTCTTGATAATTTGATAATT 8 6226 6245 2823 568328 N/A N/AACCAGGCTTTCTTGATAATT 55 6234 6253 2824 568329 N/A N/ATTTGAACCAGGCTTTCTTGA 49 6239 6258 2825 568330 N/A N/ACATAATTTGAACCAGGCTTT 68 6244 6263 109 568331 N/A N/AAGACATAATACATAATTTGA 8 6254 6273 2826 568332 N/A N/ACTGTGATAAAGACATAATAC 40 6263 6282 2827 568333 N/A N/ACAGACCTGTGATAAAGACAT 16 6268 6287 2828 568334 N/A N/AATCTTCAGACCTGTGATAAA 7 6273 6292 2829 568335 N/A N/ATACTGATCTTCAGACCTGTG 47 6278 6297 2830 568336 N/A N/ATTAATAATTTTCAGTTTTAG 35 6302 6321 2831 568337 N/A N/ATAAGTTTAATAATTTTCAGT 23 6307 6326 2832 568338 N/A N/ATTCAGATTTTAAGTTTAATA 10 6316 6335 2833 568339 N/A N/ATATATTTGATATTCTGTTCA 42 6332 6351 2834 568340 N/A N/AATATTGTAATGTATTCTTTT 0 6368 6387 2835 568341 N/A N/ATTAGAATATTGTAATGTATT 19 6373 6392 2836 568342 N/A N/ATTTGCTTAGAATATTGTAAT 9 6378 6397 2837 568343 N/A N/AACTGCTTTGCTTAGAATATT 36 6383 6402 2838 568344 N/A N/AAAGTAGAGACTGCTTTGCTT 60 6391 6410 2839 568345 N/A N/AGCAAGGCCAAAAGTAGAGAC 59 6401 6420 2840 568346 N/A N/AACAGAGCAAGGCCAAAAGTA 45 6406 6425 2841 568347 N/A N/AGGAAAACAGAGCAAGGCCAA 49 6411 6430 2842 568348 N/A N/ATGGTCGGAAAACAGAGCAAG 38 6416 6435 2843 568349 N/A N/AGACATTGGTCGGAAAACAGA 26 6421 6440 2844 568350 N/A N/AAAGCAGACATTGGTCGGAAA 50 6426 6445 2845 568351 N/A N/ACAAGGCAAAAAAGCAGACAT 39 6436 6455 2846 568352 N/A N/AATAAAGCAAGGCAAAAAAGC 20 6442 6461 2847 568353 N/A N/ACATTATTTAATAAGATAAAA 29 6464 6483 2848 568354 N/A N/AAAATATTTAATCAGGGACAT 35 6481 6500 2849 568355 N/A N/ATGTTCTCAAAATATTTAATC 32 6489 6508 2850 568356 N/A N/AGATTACCTGTTCTCAAAATA 40 6496 6515 2851 568357 N/A N/AGATTGTACAGATTACCTGTT 12 6505 6524 2852 568358 N/A N/AATTCAGATTGTACAGATTAC 34 6510 6529 2853 568359 N/A N/AAAACAGTGTTATTCAGATTG 32 6520 6539 2854 568360 N/A N/ATAGATAAACAGTGTTATTCA 25 6525 6544 2855 568361 N/A N/AATATTTAGATAAACAGTGTT 14 6530 6549 2856 568362 N/A N/AGTTTGATATTTAGATAAACA 27 6535 6554 2857 568363 N/A N/AAACGGTGTTTGATATTTAGA 33 6541 6560 2858 568364 N/A N/AGTTATAACGGTGTTTGATAT 29 6546 6565 2859 568365 N/A N/AATAATGTTATAACGGTGTTT 21 6551 6570 2860 568366 N/A N/AAGTTCATAATGTTATAACGG 37 6556 6575 2861 568367 N/A N/ACTTTCAGTTCATAATGTTAT 46 6561 6580 2862 568368 N/A N/AAGTACAGTTTGTCTTTCAGT 48 6573 6592 2863 568369 N/A N/ATCAGAAGTACAGTTTGTCTT 47 6578 6597 2864 568370 N/A N/AGGATGTCAGAAGTACAGTTT 46 6583 6602 2865 568371 N/A N/AGAGTAAGGATGTCAGAAGTA 45 6589 6608 2866 568372 N/A N/AGAAATCTGAGTAAGGATGTC 31 6596 6615 2867 568373 N/A N/ATACTGAATATACAATTAGGG 5 6616 6635 2868 568374 N/A N/AAATGATACTGAATATACAAT 21 6621 6640 2869 568375 N/A N/AGAATATAAATCTGTTTTTTA 19 6642 6661 2870 568376 N/A N/ATAAAAGAATATAAATCTGTT 32 6647 6666 2871 568377 N/A N/AGCTGATAAAAGAATATAAAT 50 6652 6671 2872 568378 N/A N/ACCTTCTGAGCTGATAAAAGA 37 6660 6679 2873 568379 N/A N/ACTAGTCCTTCTGAGCTGATA 45 6665 6684 2874 568380 N/A N/ATTACCATCATGTTTTACATT 30 6770 6789 2875 568381 N/A N/ACAAAGTGTCTTACCATCATG 24 6779 6798 2876 568382 N/A N/AAAACCCACCAAAGTGTCTTA 15 6787 6806 2877 568383 N/A N/AAGAAGGAAACCCACCAAAGT 22 6793 6812 2878 568384 N/A N/AAATAATAGCTTCAAGAAGGA 25 6806 6825 2879 568385 N/A N/AAATTTGATAATAATAGCTTC 24 6814 6833 2880 568386 N/A N/ATAGGGAATTTGATAATAATA 20 6819 6838 2881 568387 N/A N/AAAGAATAGGGAATTTGATAA 0 6824 6843 2882 568388 N/A N/AGTCCTAAGAATAGGGAATTT 45 6829 6848 2883 568389 N/A N/ATAGAACAAGTCCTAAGAATA 21 6837 6856 2884 568390 N/A N/ATTAGTCTAGAACAAGTCCTA 28 6843 6862 2885 568391 N/A N/AATCTTTTAGTCTAGAACAAG 21 6848 6867 2886 568392 N/A N/ATAACTATCTTTTAGTCTAGA 13 6853 6872 2887 568393 N/A N/AATCTCTTAACTATCTTTTAG 28 6859 6878 2888 568394 N/A N/ATGGATATCTCTTAACTATCT 48 6864 6883 2889 568395 N/A N/ATTTGATGGATATCTCTTAAC 35 6869 6888 2890 544120 707 726AGTTCTTGGTGCTCTTGGCT 80 6720 6739 15 337487 804 823 CACTTGTATGTTCACCTCTG76 7389 7408 28 568006 2014 2033 TTAATTCTGCTTCATTAGGT 53 10986 110052891 568007 2015 2034 TTTAATTCTGCTTCATTAGG 38 10987 11006 2892 5680082020 2039 CAGTATTTAATTCTGCTTCA 56 10992 11011 2893 568009 2021 2040ACAGTATTTAATTCTGCTTC 63 10993 11012 2894 568010 2022 2041TACAGTATTTAATTCTGCTT 56 10994 11013 2895 568011 2023 2042ATACAGTATTTAATTCTGCT 39 10995 11014 2896 568012 2024 2043AATACAGTATTTAATTCTGC 21 10996 11015 2897 568013 2025 2044TAATACAGTATTTAATTCTG 12 10997 11016 2898 568014 2027 2046TTTAATACAGTATTTAATTC 0 10999 11018 2899 568015 2028 2047TTTTAATACAGTATTTAATT 15 11000 11019 2900 568016 2031 2050TTATTTTAATACAGTATTTA 0 11003 11022 2901 568017 2034 2053AACTTATTTTAATACAGTAT 24 11006 11025 2902 568018 2035 2054GAACTTATTTTAATACAGTA 21 11007 11026 2903 568019 2036 2055CGAACTTATTTTAATACAGT 2 11008 11027 2904 568020 2037 2056GCGAACTTATTTTAATACAG 54 11009 11028 2905 568021 2038 2057AGCGAACTTATTTTAATACA 35 11010 11029 2906 568022 2039 2058CAGCGAACTTATTTTAATAC 50 11011 11030 2907 568023 2040 2059ACAGCGAACTTATTTTAATA 34 11012 11031 2908 568024 2041 2060GACAGCGAACTTATTTTAAT 52 11013 11032 2909 568025 2042 2061AGACAGCGAACTTATTTTAA 58 11014 11033 2910 568026 2044 2063AAAGACAGCGAACTTATTTT 32 11016 11035 2911 568027 2045 2064TAAAGACAGCGAACTTATTT 26 11017 11036 2912 568028 2048 2067GTTTAAAGACAGCGAACTTA 62 11020 11039 2913 568029 2049 2068TGTTTAAAGACAGCGAACTT 58 11021 11040 2914 568030 2050 2069TTGTTTAAAGACAGCGAACT 52 11022 11041 2915 568031 2051 2070TTTGTTTAAAGACAGCGAAC 61 11023 11042 2916 568032 2052 2071ATTTGTTTAAAGACAGCGAA 41 11024 11043 2917 568033 2053 2072CATTTGTTTAAAGACAGCGA 60 11025 11044 2918 568034 2054 2073CCATTTGTTTAAAGACAGCG 88 11026 11045 98 568035 2055 2074TCCATTTGTTTAAAGACAGC 57 11027 11046 2919 568036 2056 2075CTCCATTTGTTTAAAGACAG 58 11028 11047 2920 568037 2058 2077ATCTCCATTTGTTTAAAGAC 56 11030 11049 2921 568038 2059 2078CATCTCCATTTGTTTAAAGA 54 11031 11050 2922 568039 2060 2079TCATCTCCATTTGTTTAAAG 62 11032 11051 2923 568040 2061 2080GTCATCTCCATTTGTTTAAA 53 11033 11052 2924 568041 2063 2082TAGTCATCTCCATTTGTTTA 48 11035 11054 2925 568042 2064 2083GTAGTCATCTCCATTTGTTT 44 11036 11055 2926 568043 2065 2084AGTAGTCATCTCCATTTGTT 48 11037 11056 2927 568044 2066 2085TAGTAGTCATCTCCATTTGT 45 11038 11057 2928 568045 2067 2086TTAGTAGTCATCTCCATTTG 66 11039 11058 2929 568046 2068 2087CTTAGTAGTCATCTCCATTT 66 11040 11059 2930 568047 2069 2088ACTTAGTAGTCATCTCCATT 68 11041 11060 99 568048 2070 2089GACTTAGTAGTCATCTCCAT 77 11042 11061 100 568049 2071 2090TGACTTAGTAGTCATCTCCA 70 11043 11062 101 568050 2072 2091GTGACTTAGTAGTCATCTCC 65 11044 11063 2931 568051 2073 2092TGTGACTTAGTAGTCATCTC 49 11045 11064 2932 568052 2074 2093ATGTGACTTAGTAGTCATCT 47 11046 11065 2933 568053 2075 2094AATGTGACTTAGTAGTCATC 48 11047 11066 2934 568054 2076 2095CAATGTGACTTAGTAGTCAT 60 11048 11067 2935 568055 2077 2096TCAATGTGACTTAGTAGTCA 54 11049 11068 2936 568056 2078 2097GTCAATGTGACTTAGTAGTC 72 11050 11069 102 568057 2079 2098AGTCAATGTGACTTAGTAGT 62 11051 11070 2937 568058 2083 2102TTAAAGTCAATGTGACTTAG 15 11055 11074 2938 568059 2084 2103GTTAAAGTCAATGTGACTTA 28 11056 11075 2939 568060 2085 2104TGTTAAAGTCAATGTGACTT 35 11057 11076 2940 568061 2086 2105ATGTTAAAGTCAATGTGACT 17 11058 11077 2941 568062 2087 2106CATGTTAAAGTCAATGTGAC 27 11059 11078 2942 568063 2089 2108CTCATGTTAAAGTCAATGTG 28 11061 11080 2943 568064 2090 2109CCTCATGTTAAAGTCAATGT 50 11062 11081 2944 568066 2091 2110ACCTCATGTTAAAGTCAATG 48 11063 11082 2945 568068 2092 2111TACCTCATGTTAAAGTCAAT 13 11064 11083 2946 568069 2093 2112ATACCTCATGTTAAAGTCAA 43 11065 11084 2947 568072 2094 2113GATACCTCATGTTAAAGTCA 40 11066 11085 2948 568073 2095 2114TGATACCTCATGTTAAAGTC 40 11067 11086 2949 568075 2096 2115GTGATACCTCATGTTAAAGT 37 11068 11087 2950 568077 2097 2116AGTGATACCTCATGTTAAAG 6 11069 11088 2951 568078 2098 2117TAGTGATACCTCATGTTAAA 12 11070 11089 2952 568079 2099 2118ATAGTGATACCTCATGTTAA 8 11071 11090 2953 568080 2100 2119TATAGTGATACCTCATGTTA 13 11072 11091 2954 568081 2101 2120GTATAGTGATACCTCATGTT 41 11073 11092 2955 568082 2102 2121GGTATAGTGATACCTCATGT 53 11074 11093 2956 568083 2106 2125ATAAGGTATAGTGATACCTC 54 11078 11097 2957 568084 2107 2126AATAAGGTATAGTGATACCT 38 11079 11098 2958

TABLE 138 Inhibition of ANGPTL3 mRNA by 5-10-5 MOE gapmers targeting SEQID NO: 1 and 2 SEQ SEQ SEQ ID SEQ ID ID NO: NO: ID NO: NO: 2 SEQ ISIS 1Start 1 Stop % 2 Start Stop ID NO Site Site Sequence inhibition SiteSite NO 544120 707 726 AGTTCTTGGTGCTCTTGGCT 83 6720 6739 15 337487 804823 CACTTGTATGTTCACCTCTG 81 7389 7408 28 567295 1452 1471TAATGTTTAAATTATTGCCT 43 10424 10443 2959 567296 1455 1474GGTTAATGTTTAAATTATTG 22 10427 10446 2960 567297 1456 1475AGGTTAATGTTTAAATTATT 0 10428 10447 2961 567298 1457 1476GAGGTTAATGTTTAAATTAT 0 10429 10448 2962 567299 1458 1477TGAGGTTAATGTTTAAATTA 6 10430 10449 2963 567300 1460 1479AATGAGGTTAATGTTTAAAT 14 10432 10451 2964 567301 1461 1480GAATGAGGTTAATGTTTAAA 5 10433 10452 2965 567302 1462 1481GGAATGAGGTTAATGTTTAA 27 10434 10453 2966 567303 1463 1482TGGAATGAGGTTAATGTTTA 32 10435 10454 2967 567304 1464 1483TTGGAATGAGGTTAATGTTT 37 10436 10455 2968 567305 1465 1484CTTGGAATGAGGTTAATGTT 25 10437 10456 2969 567306 1468 1487TAACTTGGAATGAGGTTAAT 29 10440 10459 2970 567307 1469 1488TTAACTTGGAATGAGGTTAA 44 10441 10460 2971 337513 1470 1489ATTAACTTGGAATGAGGTTA 52 10442 10461 2972 567308 1471 1490CATTAACTTGGAATGAGGTT 62 10443 10462 2973 567309 1472 1491ACATTAACTTGGAATGAGGT 58 10444 10463 2974 567310 1473 1492CACATTAACTTGGAATGAGG 78 10445 10464 92 567311 1475 1494ACCACATTAACTTGGAATGA 59 10447 10466 2975 567312 1476 1495GACCACATTAACTTGGAATG 57 10448 10467 2976 337514 1477 1496AGACCACATTAACTTGGAAT 71 10449 10468 2977 567313 1478 1497TAGACCACATTAACTTGGAA 43 10450 10469 2978 567314 1479 1498TTAGACCACATTAACTTGGA 59 10451 10470 2979 567315 1480 1499ATTAGACCACATTAACTTGG 70 10452 10471 2980 567316 1481 1500TATTAGACCACATTAACTTG 53 10453 10472 2981 567317 1482 1501TTATTAGACCACATTAACTT 49 10454 10473 2982 567318 1483 1502ATTATTAGACCACATTAACT 41 10455 10474 2983 567319 1484 1503GATTATTAGACCACATTAAC 47 10456 10475 2984 567320 1487 1506CCAGATTATTAGACCACATT 86 10459 10478 93 567321 1489 1508TACCAGATTATTAGACCACA 85 10461 10480 94 337516 1490 1509ATACCAGATTATTAGACCAC 77 10462 10481 86 567322 1491 1510AATACCAGATTATTAGACCA 50 10463 10482 2985 567323 1492 1511TAATACCAGATTATTAGACC 56 10464 10483 2986 567324 1494 1513TTTAATACCAGATTATTAGA 9 10466 10485 2987 567325 1495 1514ATTTAATACCAGATTATTAG 24 10467 10486 2988 567326 1496 1515GATTTAATACCAGATTATTA 37 10468 10487 2989 567327 1500 1519TAAGGATTTAATACCAGATT 60 10472 10491 2990 567328 1507 1526TTTCTCTTAAGGATTTAATA 34 10479 10498 2991 567329 1508 1527CTTTCTCTTAAGGATTTAAT 46 10480 10499 2992 567330 1509 1528GCTTTCTCTTAAGGATTTAA 75 10481 10500 95 567331 1510 1529AGCTTTCTCTTAAGGATTTA 59 10482 10501 2993 567332 1511 1530AAGCTTTCTCTTAAGGATTT 30 10483 10502 2994 567333 1513 1532TCAAGCTTTCTCTTAAGGAT 65 10485 10504 2995 567334 1514 1533CTCAAGCTTTCTCTTAAGGA 77 10486 10505 96 567335 1515 1534TCTCAAGCTTTCTCTTAAGG 75 10487 10506 97 567336 1516 1535TTCTCAAGCTTTCTCTTAAG 59 10488 10507 2996 567337 1517 1536TTTCTCAAGCTTTCTCTTAA 52 10489 10508 2997 567338 1521 1540TCTATTTCTCAAGCTTTCTC 68 10493 10512 2998 567339 1522 1541ATCTATTTCTCAAGCTTTCT 71 10494 10513 2999 567340 1523 1542AATCTATTTCTCAAGCTTTC 74 10495 10514 3000 567341 1524 1543AAATCTATTTCTCAAGCTTT 63 10496 10515 3001 567342 1525 1544AAAATCTATTTCTCAAGCTT 54 10497 10516 3002 567343 1532 1551GATAAAAAAAATCTATTTCT 30 10504 10523 3003 567344 1548 1567TAGACAGTGACTTTAAGATA 37 10520 10539 3004 567345 1549 1568ATAGACAGTGACTTTAAGAT 29 10521 10540 3005 567346 1550 1569AATAGACAGTGACTTTAAGA 48 10522 10541 3006 567347 1551 1570AAATAGACAGTGACTTTAAG 26 10523 10542 3007 567348 1552 1571TAAATAGACAGTGACTTTAA 26 10524 10543 3008 567349 1553 1572TTAAATAGACAGTGACTTTA 50 10525 10544 3009 567350 1554 1573CTTAAATAGACAGTGACTTT 63 10526 10545 3010 567351 1555 1574TCTTAAATAGACAGTGACTT 57 10527 10546 3011 567352 1556 1575ATCTTAAATAGACAGTGACT 69 10528 10547 3012 567353 1557 1576AATCTTAAATAGACAGTGAC 40 10529 10548 3013 567354 1558 1577TAATCTTAAATAGACAGTGA 30 10530 10549 3014 567355 1559 1578TTAATCTTAAATAGACAGTG 25 10531 10550 3015 567356 1560 1579TTTAATCTTAAATAGACAGT 0 10532 10551 3016 567357 1561 1580GTTTAATCTTAAATAGACAG 34 10533 10552 3017 567358 1562 1581TGTTTAATCTTAAATAGACA 5 10534 10553 3018 567359 1563 1582ATGTTTAATCTTAAATAGAC 0 10535 10554 3019 567360 1567 1586TTGTATGTTTAATCTTAAAT 0 10539 10558 3020 567361 1568 1587ATTGTATGTTTAATCTTAAA 8 10540 10559 3021 567362 1569 1588GATTGTATGTTTAATCTTAA 20 10541 10560 3022 567363 1570 1589TGATTGTATGTTTAATCTTA 29 10542 10561 3023 567364 1574 1593TATGTGATTGTATGTTTAAT 7 10546 10565 3024 567365 1576 1595GTTATGTGATTGTATGTTTA 43 10548 10567 3025 567366 1580 1599TAAGGTTATGTGATTGTATG 28 10552 10571 3026 567367 1581 1600TTAAGGTTATGTGATTGTAT 31 10553 10572 3027 567368 1585 1604TTCTTTAAGGTTATGTGATT 12 10557 10576 3028

Example 117: Dose-Dependent Antisense Inhibition of Human ANGPTL3 inHep3B Cells by MOE Gapmers

5-10-5 MOE gapmers from the studies described above exhibitingsignificant in vitro inhibition of ANGPTL3 mRNA were selected and testedat various doses in Hep3B cells. Cells were plated at a density of20,000 cells per well and transfected using electroporation with 0.75μM, 1.50 μM, 3.00 μM, 6.00 μM and 12.00 μM concentrations of antisenseoligonucleotide, as specified in the Table below. After a treatmentperiod of approximately 16 hours, RNA was isolated from the cells andANGPTL3 mRNA levels were measured by quantitative real-time PCR. Humanprimer probe set RTS3492_MGB was used to measure mRNA levels. ANGPTL3mRNA levels were adjusted according to total RNA content, as measured byRIBOGREEN. Results are presented as percent inhibition of ANGPTL3,relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented. ANGPTL3 mRNA levels were significantly reduced in adose-dependent manner in antisense oligonucleotide treated cells.

TABLE 139 0.75 3.00 12.00 IC₅₀ SEQ ID ISIS No μM 1.50 μM μM 6.00 μM μM(μM) NO 233717 23 45 13 33 40 >12 14 544120 45 65 76 88 91 0.7 15 54414538 42 61 82 84 1.6 16 544156 31 42 63 78 84 1.8 17 544162 35 43 71 76 821.6 18 544166 30 47 60 76 84 1.8 19 544199 54 61 73 83 84 0.5 20 54435545 46 69 77 83 1.2 21 544368 12 37 63 74 81 2.6 22 544373 1 27 40 2928 >12 23 544376 26 53 61 63 59 2.4 24 544380 16 33 41 64 39 8.4 25544383 14 33 46 61 63 4.4 26 544410 10 41 48 62 69 3.6 27

Example 118: Dose-Dependent Antisense Inhibition of Human ANGPTL3 inHep3B Cells by MOE Gapmers

5-10-5 MOE gapmers from the studies described above exhibitingsignificant in vitro inhibition of ANGPTL3 mRNA were selected and testedat various doses in Hep3B cells. Cells were plated at a density of20,000 cells per well and transfected using electroporation with 0.813μM, 1.625 μM, 3.25 μM, 6.50 μM and 13.00 μM concentrations of antisenseoligonucleotide, as specified in the Table below. After a treatmentperiod of approximately 16 hours, RNA was isolated from the cells andANGPTL3 mRNA levels were measured by quantitative real-time PCR. Humanprimer probe set RTS3492_MGB was used to measure mRNA levels. ANGPTL3mRNA levels were adjusted according to total RNA content, as measured byRIBOGREEN®. Results are presented as percent inhibition of ANGPTL3,relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented. ANGPTL3 mRNA levels were significantly reduced in adose-dependent manner in antisense oligonucleotide treated cells.

TABLE 140 SEQ 0.813 1.625 IC₅₀ ID ISIS No μM μM 3.25 μM 6.50 μM 13.00 μM(μM) NO 337487 17 37 58 72 92 2.7 28 337492 0 0 0 5 58 >13 29 544120 2340 65 81 91 2.2 15 560236 39 22 46 9 60 >13 30 560265 38 48 58 64 73 2.031 560268 37 57 60 71 83 1.5 32 560285 5 29 48 68 78 3.8 33 560306 45 6467 81 86 0.9 34 560400 48 63 75 87 88 0.7 35 560401 49 75 79 89 88 0.536 560402 42 67 70 85 90 0.9 37 560469 43 55 70 74 83 1.2 38 560470 3154 64 73 81 1.8 39 560471 26 43 59 62 77 2.7 40 560474 42 50 60 54 721.8 41

TABLE 141 SEQ 0.813 1.625 IC₅₀ ID ISIS No μM μM 3.25 μM 6.50 μM 13.00 μM(μM) NO 337487 20 35 51 78 89 1.8 28 544120 31 46 62 84 90 0.5 15 5441454 36 60 58 89 3.8 16 544156 22 35 46 66 73 1.8 17 544162 2 21 54 6987 >13 18 544166 15 0 25 59 89 >13 19 544199 61 37 57 53 81 0.9 20544355 0 47 50 73 84 >13 21 544376 4 14 38 66 88 0.9 24 560566 53 68 7076 85 >13 42 560567 55 70 75 78 89 2.7 43 560574 49 63 68 74 84 2.0 44560596 28 40 41 52 75 1.5 45 560607 35 53 65 70 85 3.8 46 560608 40 5062 68 83 0.9 47 560723 36 51 59 65 75 2.2 48 560735 36 44 59 72 85 >1349 560736 26 34 50 64 80 0.7 50 560744 28 49 59 75 83 0.9 51 560778 2446 60 67 85 1.8 52 560789 14 23 36 49 71 2.7 53 560811 32 50 65 73 871.2 54 560856 0 20 17 32 69 3.8 55 560925 2 16 38 52 82 2.7 56 560936 00 24 41 65 0.5 57 560938 0 26 30 43 50 0.9 58 560942 0 0 12 36 74 1.8 59560956 0 16 16 68 81 0.5 60

TABLE 142 IC₅₀ SEQ ID ISIS No 0.813 μM 1.625 μM 3.25 μM 6.50 μM 13.00 μM(μM) NO 337487 20 35 51 78 89 2.7 28 544120 31 46 62 84 90 1.9 15 56056653 68 70 76 85 0.5 42 560567 55 70 75 78 89 0.4 43 560574 49 63 68 74 840.7 44 560596 28 40 41 52 75 3.9 45 560607 35 53 65 70 85 1.6 46 56060840 50 62 68 83 1.6 47 560723 36 51 59 65 75 1.9 48 560735 36 44 59 72 852.0 49 560736 26 34 50 64 80 3.2 50 560744 28 49 59 75 83 2.1 51 56077824 46 60 67 85 2.4 52 560789 14 23 36 49 71 5.7 53 560811 32 50 65 73 871.8 54

TABLE 143 IC₅₀ SEQ ID ISIS No 0.813 μM 1.625 μM 3.25 μM 6.50 μM 13.00 μM(μM) NO 337487 10 21 49 73 90 3.4 28 544120 19 38 62 77 88 2.5 15 5607681 14 14 28 51 >13 61 560777 13 35 37 56 80 4.2 62 560791 13 28 28 2411 >13 63 560794 8 31 42 57 76 4.4 64 560799 0 14 21 43 72 7.2 65 56080326 44 52 55 69 3.4 66 560815 16 26 26 52 60 7.6 67 560817 0 0 11 1837 >13 68 560847 37 52 56 68 87 1.8 69 560879 15 18 38 53 72 5.4 70560880 0 8 21 38 71 8.0 71 560891 7 25 32 35 62 8.9 72 560895 11 10 0 548 >13 73

TABLE 144 IC₅₀ SEQ ID ISIS No 0.813 μM 1.625 μM 3.25 μM 6.50 μM 13.00 μM(μM) NO 337487 20 14 38 65 88 3.9 28 544120 22 34 51 71 86 2.9 15 54414521 39 62 63 90 2.6 16 544156 31 41 55 72 78 2.4 17 544162 0 37 59 75 872.7 18 544166 8 43 45 55 75 4.0 19 544199 53 46 64 62 81 1.1 20 544355 00 52 72 84 2.9 21 544376 2 22 39 51 76 5.2 24 560856 10 29 36 41 69 6.455 560925 0 35 46 59 81 3.5 56 560936 18 9 35 55 69 5.9 57 560938 14 3442 49 58 6.5 58 560942 8 13 27 47 77 6.1 59 560956 16 31 0 69 81 3.9 60

TABLE 145 IC₅₀ SEQ ID ISIS No 0.813 μM 1.625 μM 3.25 μM 6.50 μM 13.00 μM(μM) NO 233717 11 0 33 58 75 5.0 14 337484 39 54 55 66 79 1.7 74 33748735 42 67 82 92 1.8 28 544120 53 47 78 84 92 <0.8 15 563523 12 44 59 6379 3.0 75 563547 33 51 55 43 58 4.6 76 563580 61 73 71 82 91 <0.8 77563637 36 55 69 77 88 1.4 78 563639 56 71 79 88 93 <0.8 79 563641 30 4256 77 84 2.2 80 563669 28 61 66 79 85 1.6 81 563681 35 67 74 75 70 0.982 563682 41 45 68 76 85 1.5 83 567068 32 37 50 66 81 2.8 84 567069 2328 48 56 62 5.0 85

TABLE 146 IC₅₀ SEQ ID ISIS No 0.813 μM 1.625 μM 3.25 μM 6.50 μM 13.00 μM(μM) NO 233717 9 0 25 62 74 5.5 14 337487 22 40 71 84 92 2.1 28 33751636 54 78 81 92 1.3 86 544120 25 50 72 86 92 1.8 15 567078 54 64 70 78 78<0.8 87 567115 55 65 72 80 81 <0.8 88 567134 33 58 53 57 69 2.2 89567233 54 74 83 87 91 <0.8 90 567291 54 67 71 80 89 <0.8 91 567310 36 6173 80 89 1.2 92 567320 63 77 88 88 92 <0.8 93 567321 55 75 89 89 93 <0.894 567330 31 68 76 85 93 1.2 95 567334 36 54 76 82 87 1.3 96 567335 3149 72 80 92 1.7 97

TABLE 147 IC₅₀ SEQ ID ISIS No 0.813 μM 1.625 μM 3.25 μM 6.50 μM 13.00 μM(μM) NO 233717 0 0 23 66 64 6.6 14 337487 13 44 60 74 85 2.6 28 54412024 47 53 78 83 2.3 15 568034 35 54 51 59 46 4.2 98 568047 36 55 70 69 721.4 99 568048 41 64 63 66 66 0.9 100 568049 50 70 70 74 73 <0.8 101568056 33 56 68 63 64 1.7 102 568144 27 57 63 63 76 2.0 103 568146 50 6161 63 77 <0.8 104 568151 23 46 59 68 66 2.8 105 568206 24 40 56 61 753.0 106 568293 0 39 46 59 78 4.1 107 568300 22 36 61 68 73 3.0 108568330 16 48 54 73 82 2.7 109

Example 119: Antisense Inhibition of Human ANGPTL3 in Hep3B Cells byDeoxy, MOE and (S)-cEt Gapmers

Additional antisense oligonucleotides were designed targeting an ANGPTL3nucleic acid and were tested for their effects on ANGPTL3 mRNA in vitro.Cultured Hep3B cells at a density of 20,000 cells per well weretransfected using electroporation with 4,500 nM of antisenseoligonucleotide. After a treatment period of approximately 24 hours, RNAwas isolated from the cells and ANGPTL3 mRNA levels were measured byquantitative real-time PCR. Human primer probe set RTS3492_MGB was usedto measure mRNA levels. ANGPTL3 mRNA levels were adjusted according tototal RNA content, as measured by RIBOGREEN®. Results are presented aspercent inhibition of ANGPTL3, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in the Tablesbelow were designed as deoxy, MOE, and (S)-cEt oligonucleotides. Thedeoxy, MOE and (S)-cEt oligonucleotides are 16 nucleosides in lengthwherein the nucleoside have either a MOE sugar modification, a (S)-cEtsugar modification, or a deoxy sugar residue. The sugar modifications ofeach antisense oligonucleotide is described as ‘eek-d 10-kke’, where ‘k’indicates a (S)-cEt sugar modification; ‘d’ indicates deoxyribose; thenumber indicates the number of deoxyribose sugars residues; and ‘e’indicates a MOE sugar modification. The internucleoside linkagesthroughout each oligonucleotide are phosphorothioate (P═S) linkages. Allcytosine residues throughout each oligonucleotide are 5-methylcytosines.“Start site” indicates the 5′-most nucleoside to which theoligonucleotide is targeted in the human gene sequence. “Stop site”indicates the 3′-most nucleoside to which the oligonucleotide istargeted human gene sequence. Each oligonucleotide listed in the Tablesbelow is targeted to either the human ANGPTL3 mRNA, designated herein asSEQ ID NO: 1 (GENBANK Accession No. NM_014495.2) or the human ANGPTL3genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK AccessionNo. NT_032977.9 truncated from nucleotides 33032001 to 33046000). ‘n/a’indicates that the antisense oligonucleotide does not target thatparticular gene sequence with 100% complementarity.

TABLE 148 Inhibition of ANGPTL3 mRNA by deoxy, MOE and cEtoligonucleotides targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID IDID NO: 1 NO: NO: 2 NO: 2 SEQ ISIS Start 1 Stop % Start Stop ID NO SiteSite Sequence inhibition Site Site NO 561681 N/A N/A TCTGGAAGCAGACCTA 373096 3111 3029 561682 N/A N/A CTTCTGGAAGCAGACC 27 3098 3113 3030 561683N/A N/A AAATAAGGTATAGTGA 2 11084 11099 3031 561684 N/A N/ATAGTATTAAGTGTTAA 14 11133 11148 3032 561685 N/A N/A TCATAGTATTAAGTGT 011136 11151 3033 561686 N/A N/A AGATTCCTTTACAATT 21 11160 11175 3034561687 N/A N/A ACAAGATTCCTTTACA 21 11163 11178 3035 561688 N/A N/ACTGACAAGATTCCTTT 70 11166 11181 3036 561689 N/A N/A AATCTGACAAGATTCC 8311169 11184 180 561690 N/A N/A TGTAATCTGACAAGAT 46 11172 11187 3037561691 N/A N/A TACTGTAATCTGACAA 47 11175 11190 3038 561692 N/A N/ATCTTACTGTAATCTGA 50 11178 11193 3039 561693 N/A N/A CATTCTTACTGTAATC 4011181 11196 3040 561694 N/A N/A GTTCATTCTTACTGTA 71 11184 11199 3041561695 N/A N/A ATATGTTCATTCTTAC 2 11188 11203 3042 561696 N/A N/AGCCACAAATATGTTCA 80 11195 11210 3043 561697 N/A N/A GATGCCACAAATATGT 7011198 11213 3044 561698 N/A N/A CTCGATGCCACAAATA 80 11201 11216 181561699 N/A N/A TAACTCGATGCCACAA 86 11204 11219 182 561700 N/A N/ACTTTAACTCGATGCCA 77 11207 11222 3045 561701 N/A N/A AAACTTTAACTCGATG 3911210 11225 3046 561702 N/A N/A TATAAACTTTAACTCG 13 11213 11228 3047561703 N/A N/A CACAGCATATTTAGGG 71 11233 11248 3048 561704 N/A N/ATAGAATCACAGCATAT 68 11239 11254 3049 561705 N/A N/A TATTAGAATCACAGCA 7311242 11257 3050 561706 N/A N/A AATGTATTAGAATCAC 40 11246 11261 3051561707 N/A N/A ACGAATGTATTAGAAT 22 11249 11264 3052 561708 N/A N/ATACACGAATGTATTAG 33 11252 11267 3053 561709 N/A N/A ACCTACACGAATGTAT 4211255 11270 3054 561710 N/A N/A AAAACCTACACGAATG 24 11258 11273 3055561711 N/A N/A TTGAAAACCTACACGA 34 11261 11276 3056 561712 N/A N/ATACTTGAAAACCTACA 33 11264 11279 3057 561713 N/A N/A GTTTATTTCTACTTGA 5311273 11288 3058 561714 N/A N/A GAGGTTTATTTCTACT 69 11276 11291 3059561715 N/A N/A TACGAGGTTTATTTCT 21 11279 11294 3060 561716 N/A N/ATGTTACGAGGTTTATT 47 11282 11297 3061 561717 N/A N/A ACTTGTTACGAGGTTT 7011285 11300 3062 561718 N/A N/A CAGTAACTTGTTACGA 60 11290 11305 3063561719 N/A N/A GTTCAGTAACTTGTTA 40 11293 11308 3064 561720 N/A N/ATCAGGCTGTTTAAACG 59 11308 11323 3065 561721 N/A N/A TTGTCAGGCTGTTTAA 7411311 11326 3066 561722 N/A N/A TGCTTGTCAGGCTGTT 82 11314 11329 183561723 N/A N/A ACATGCTTGTCAGGCT 84 11317 11332 184 561724 N/A N/ATATACATGCTTGTCAG 75 11320 11335 3067 561725 N/A N/A GTCTTTGTTTATTGAA 4911347 11362 3068 561726 N/A N/A TGGGTCTTTGTTTATT 27 11350 11365 3069561727 N/A N/A GACTGGGTCTTTGTTT 20 11353 11368 3070 561728 N/A N/AATAATTTAGGGACTGG 20 11363 11378 3071 561729 N/A N/A TCTATAATTTAGGGAC 3911366 11381 3072 561730 N/A N/A CGATAAACATGCAAGA 68 11394 11409 3073561731 N/A N/A TGTCGATAAACATGCA 80 11397 11412 3074 561732 N/A N/ATGATGTCGATAAACAT 68 11400 11415 3075 561733 N/A N/A TTGTGATGTCGATAAA 2811403 11418 3076 561734 N/A N/A CTGTTGTGATGTCGAT 74 11406 11421 3077561735 N/A N/A GATCTGTTGTGATGTC 59 11409 11424 3078 561736 N/A N/AAGGGATCTGTTGTGAT 24 11412 11427 3079 561737 N/A N/A TTTAGGGATCTGTTGT 1911415 11430 3080 561738 N/A N/A GGATTTAGGGATCTGT 27 11418 11433 3081561739 N/A N/A GATTTAGGGATTTAGG 44 11425 11440 3082 561740 N/A N/ATCTTTAGGGATTTAGG 38 11433 11448 3083 561741 N/A N/A TAATCTTTAGGGATTT 011436 11451 3084 561742 N/A N/A ATCTAATCTTTAGGGA 0 11439 11454 3085561743 N/A N/A TGTATCTAATCTTTAG 15 11442 11457 3086 561744 N/A N/AAAATTTGTATCTAATC 21 11447 11462 3087 561745 N/A N/A GTAAAAAATTTGTATC 2311452 11467 3088 561746 N/A N/A GTGGTAAAAAATTTGT 32 11455 11470 3089561747 N/A N/A GATACTGTGGTAAAAA 45 11461 11476 3090 561748 N/A N/AAGTGATACTGTGGTAA 60 11464 11479 3091 561749 N/A N/A ACAAGTGATACTGTGG 7511467 11482 3092 561750 N/A N/A CTGACAAGTGATACTG 59 11470 11485 3093561751 N/A N/A ATTCTGACAAGTGATA 48 11473 11488 3094 561752 N/A N/ATAAATTCTGACAAGTG 59 11476 11491 3095 561753 N/A N/A TACTGGCAGTTTTAAA 4211508 11523 3096 561754 N/A N/A TCTTACTGGCAGTTTT 51 11511 11526 3097561755 N/A N/A ATTTCTTACTGGCAGT 69 11514 11529 3098 561756 N/A N/AAAAATTTCTTACTGGC 57 11517 11532 3099 561757 N/A N/A AACAAATGGGTTTAAT 011535 11550 3100 562374 N/A N/A GAATATTTGCAAGTCT 68 9230 9245 3101562375 N/A N/A GTAGAGGAATATTTGC 83 9236 9251 151 562376 N/A N/ATCATTGGTAGAGGAAT 23 9242 9257 3102 562377 N/A N/A ATATTTTAAAGTCTCG 179258 9273 3103 562378 N/A N/A GTTACATTATTATAGA 29 9273 9288 3104 562379N/A N/A GTGAAATGTGTTACAT 54 9282 9297 3105 562380 N/A N/ATCACCAGTGAAATGTG 64 9288 9303 3106 562381 N/A N/A CATGTTTCACCAGTGA 789294 9309 3107 562382 N/A N/A ACAAGACATGTTTCAC 36 9300 9315 3108 562383N/A N/A CATATGACAAGACATG 42 9306 9321 3109 562384 N/A N/ACTATAATGCATATGAC 5 9314 9329 3110 562385 N/A N/A TCCTTTCTATAATGCA 659320 9335 3111 562386 N/A N/A TGATTATCCTTTCTAT 27 9326 9341 3112 562387N/A N/A AAAGTCTGATTATCCT 90 9332 9347 152 562388 N/A N/ATAACTGAAAGTCTGAT 59 9338 9353 3113 562389 N/A N/A GTGCACAAAAATGTTA 429366 9381 3114 562390 N/A N/A AGCTATGTGCACAAAA 77 9372 9387 3115 562391N/A N/A GAAGATAGCTATGTGC 64 9378 9393 3116 562392 N/A N/ATTTATTGAAGATAGCT 33 9384 9399 3117 562393 N/A N/A TCATTTTAGTGTATCT 409424 9439 3118 562394 N/A N/A CCTTGATCATTTTAGT 15 9430 9445 3119 562395N/A N/A TGAATCCCTTGATCAT 59 9436 9451 3120 562396 N/A N/ATAGTCTTGAATCCCTT 83 9442 9457 153 562397 N/A N/A GTTGTTTAGTCTTGAA 659448 9463 3121 562398 N/A N/A AATTGAGTTGTTTAGT 21 9454 9469 3122 562399N/A N/A GCAACTAATTGAGTTG 15 9460 9475 3123 562400 N/A N/AATTGGTGCAACTAATT 25 9466 9481 3124 562401 N/A N/A GTTTTTTATTGGTGCA 539473 9488 3125 562402 N/A N/A GGACACTGACAGTTTT 43 9496 9511 3126 562403N/A N/A CAGGTTGGACACTGAC 23 9502 9517 3127 562404 N/A N/ATAAGTACAGGTTGGAC 33 9508 9523 3128 562405 N/A N/A AGTTATTAAGTACAGG 349514 9529 3129 562406 N/A N/A TCTGTGAGTTATTAAG 10 9520 9535 3130 562407N/A N/A ACCAAAATTCTCCTGA 1 9554 9569 3131 562408 N/A N/AACCTGAATAACCCTCT 73 9811 9826 3132 562409 N/A N/A GGTATCAGAAAAAGAT 149827 9842 3133 562410 N/A N/A AGTATTGGTATCAGAA 13 9833 9848 3134 562411N/A N/A GGAAGATACTTTGAAG 25 9861 9876 3135 562412 N/A N/AAATGTGGGAAGATACT 23 9867 9882 3136 562413 N/A N/A CAGATAATAGCTAATA 299882 9897 3137 562414 N/A N/A TCATTGCAGATAATAG 45 9888 9903 3138 562415N/A N/A AAGTTGTCATTGCAGA 86 9894 9909 154 562416 N/A N/AGATTCGGATTTTTAAA 19 9909 9924 3139 562417 N/A N/A ATTTGGGATTCGGATT 349915 9930 3140 562418 N/A N/A ACGCTTATTTGGGATT 64 9921 9936 3141 562419N/A N/A TCTAGAGAGAAAACGC 64 9933 9948 3142 562420 N/A N/AAGTTAAGAGGTTTTCG 34 9949 9964 3143 562421 N/A N/A CATTATAGTTAAGAGG 249955 9970 3144 562422 N/A N/A CACTTTCATTATAGTT 13 9961 9976 3145 562423N/A N/A TAGAATGAACACTTTC 63 9970 9985 3146 562424 N/A N/ATTGAACTAGAATGAAC 16 9976 9991 3147 562425 N/A N/A ACCTGATTGAACTAGA 519982 9997 3148 562426 N/A N/A TAAAATACCTGATTGA 19 9988 10003 3149 562427N/A N/A TAGAGGTAAAATACCT 12 9994 10009 3150 562428 N/A N/AGAAGATTAGAGGTAAA 1 10000 10015 3151 562429 N/A N/A TCTGAGGAAGATTAGA 3110006 10021 3152 562430 N/A N/A TATACACTACCAAAAA 0 10030 10045 3153562431 N/A N/A ATAATCTATACACTAC 0 10036 10051 3154 562432 N/A N/ATAAGTCCCAATTTTAA 33 10065 10080 3155 562433 N/A N/A TCTGTATAAGTCCCAA 8910071 10086 155 562434 N/A N/A CCAGTTTTAAATAATC 20 10085 10100 3156562435 N/A N/A TGTATCCCAGTTTTAA 44 10091 10106 3157 562436 N/A N/AGATGCATGTATCCCAG 91 10097 10112 156 562437 N/A N/A GTTTTAGATGCATGTA 6910103 10118 3158 562438 N/A N/A TACAGTGTTTTAGATG 28 10109 10124 3159562439 N/A N/A GTAAGTTTATCTTCCT 78 10138 10153 157 562440 N/A N/ATTCCCCGTAAGTTTAT 33 10144 10159 3160 562441 N/A N/A CTGTATTTCCCCGTAA 5510150 10165 3161 562442 N/A N/A CTGTTACTGTATTTCC 79 10156 10171 158562443 N/A N/A TAGTTACTGTTACTGT 70 10162 10177 3162 562444 N/A N/ACGTATGTAGTTACTGT 66 10168 10183 3163 562445 N/A N/A AATGGGTACAGACTCG 7210182 10197 3164 562446 N/A N/A GCAATTTAATGGGTAC 59 10189 10204 3165562447 N/A N/A GATAGATATGCAATTT 20 10198 10213 3166 562448 N/A N/AAAAGGAGATAGATATG 22 10204 10219 3167 562449 N/A N/A CCTCCTAAAGGAGATA 4210210 10225 3168 562450 N/A N/A CACCAGCCTCCTAAAG 37 10216 10231 3169560990 709 724 TTCTTGGTGCTCTTGG 89 6722 6737 111 561373 1197 1212TTTGTGATCCCAAGTA 40 9772 9787 3170 561374 1199 1214 GCTTTGTGATCCCAAG 769774 9789 3171 561375 1201 1216 TTGCTTTGTGATCCCA 82 9776 9791 3172561376 1203 1218 TTTTGCTTTGTGATCC 40 9778 9793 3173 561377 1205 1220CCTTTTGCTTTGTGAT 38 9780 9795 3174 561378 1207 1222 GTCCTTTTGCTTTGTG 759782 9797 3175 561379 1209 1224 GTGTCCTTTTGCTTTG 40 9784 9799 3176561527 1604 1619 GAAATGTAAACGGTAT 47 10576 10591 3177 561528 1606 1621GAGAAATGTAAACGGT 89 10578 10593 174 561529 1608 1623 TTGAGAAATGTAAACG 5510580 10595 3178 561530 1611 1626 TGATTGAGAAATGTAA 18 10583 10598 3179561531 1613 1628 TTTGATTGAGAAATGT 30 10585 10600 3180 561532 1619 1634AAGAATTTTGATTGAG 53 10591 10606 3181 561533 1621 1636 ATAAGAATTTTGATTG29 10593 10608 3182 561534 1632 1647 CAAATAGTATTATAAG 6 10604 10619 3183561535 1653 1668 CCCACATCACAAAATT 70 10625 10640 3184 561536 1657 1672GATTCCCACATCACAA 77 10629 10644 3185 561537 1659 1674 TTGATTCCCACATCAC78 10631 10646 3186 561538 1661 1676 AATTGATTCCCACATC 68 10633 106483187 561539 1663 1678 AAAATTGATTCCCACA 72 10635 10650 3188 561540 16651680 CTAAAATTGATTCCCA 54 10637 10652 3189 561541 1668 1683CATCTAAAATTGATTC 0 10640 10655 3190 561542 1670 1685 ACCATCTAAAATTGAT 3510642 10657 3191 561543 1672 1687 TGACCATCTAAAATTG 55 10644 10659 3192561544 1674 1689 TGTGACCATCTAAAAT 56 10646 10661 3193 561545 1676 1691ATTGTGACCATCTAAA 73 10648 10663 3194 561546 1678 1693 AGATTGTGACCATCTA67 10650 10665 3195 561547 1680 1695 CTAGATTGTGACCATC 50 10652 106673196 561548 1682 1697 ATCTAGATTGTGACCA 77 10654 10669 3197 561549 16841699 TAATCTAGATTGTGAC 55 10656 10671 3198 561550 1686 1701TATAATCTAGATTGTG 28 10658 10673 3199 561551 1688 1703 ATTATAATCTAGATTG52 10660 10675 3200 561552 1690 1705 TGATTATAATCTAGAT 43 10662 106773201 561553 1692 1707 ATTGATTATAATCTAG 53 10664 10679 3202 561554 16941709 CTATTGATTATAATCT 54 10666 10681 3203 561555 1696 1711ACCTATTGATTATAAT 44 10668 10683 3204 561556 1698 1713 TCACCTATTGATTATA52 10670 10685 3205 561557 1700 1715 GTTCACCTATTGATTA 50 10672 106873206 561558 1702 1717 AAGTTCACCTATTGAT 58 10674 10689 3207 561559 17041719 ATAAGTTCACCTATTG 66 10676 10691 3208 561560 1706 1721TAATAAGTTCACCTAT 38 10678 10693 3209 561561 1708 1723 TTTAATAAGTTCACCT50 10680 10695 3210 561562 1710 1725 TATTTAATAAGTTCAC 32 10682 106973211 561563 1712 1727 GTTATTTAATAAGTTC 47 10684 10699 3212 561564 17611776 CATATGATGCCTTTTA 63 10733 10748 3213 561565 1763 1778CTCATATGATGCCTTT 81 10735 10750 175 561566 1765 1780 AGCTCATATGATGCCT 8110737 10752 176 561567 1767 1782 TTAGCTCATATGATGC 84 10739 10754 177561568 1769 1784 TATTAGCTCATATGAT 46 10741 10756 3214 561569 1771 1786GATATTAGCTCATATG 49 10743 10758 3215 561570 1773 1788 GTGATATTAGCTCATA81 10745 10760 3216 561571 1775 1790 TTGTGATATTAGCTCA 85 10747 10762 178561572 1777 1792 AGTTGTGATATTAGCT 68 10749 10764 3217 561573 1779 1794AAAGTTGTGATATTAG 45 10751 10766 3218 561574 1781 1796 GGAAAGTTGTGATATT27 10753 10768 3219 561575 1783 1798 TGGGAAAGTTGTGATA 36 10755 107703220 561576 1785 1800 ACTGGGAAAGTTGTGA 83 10757 10772 179 561577 17871802 AAACTGGGAAAGTTGT 56 10759 10774 3221 561578 1789 1804TTAAACTGGGAAAGTT 44 10761 10776 3222 561579 1794 1809 GTTTTTTAAACTGGGA58 10766 10781 3223 561580 1796 1811 TAGTTTTTTAAACTGG 0 10768 10783 3224561581 1802 1817 GAGTACTAGTTTTTTA 18 10774 10789 3225 561582 1804 1819AAGAGTACTAGTTTTT 55 10776 10791 3226 561583 1806 1821 ACAAGAGTACTAGTTT51 10778 10793 3227 561584 1808 1823 TAACAAGAGTACTAGT 53 10780 107953228 561585 1810 1825 TTTAACAAGAGTACTA 48 10782 10797 3229 561586 18121827 GTTTTAACAAGAGTAC 49 10784 10799 3230 561587 1814 1829GAGTTTTAACAAGAGT 54 10786 10801 3231 561588 1816 1831 TAGAGTTTTAACAAGA 910788 10803 3232 561589 1819 1834 GTTTAGAGTTTTAACA 24 10791 10806 3233561590 1822 1837 CAAGTTTAGAGTTTTA 30 10794 10809 3234 561591 1824 1839GTCAAGTTTAGAGTTT 60 10796 10811 3235 561592 1826 1841 TAGTCAAGTTTAGAGT56 10798 10813 3236 561593 1828 1843 TTTAGTCAAGTTTAGA 41 10800 108153237 561594 1830 1845 TATTTAGTCAAGTTTA 14 10802 10817 3238 561595 18321847 TGTATTTAGTCAAGTT 39 10804 10819 3239 561596 1834 1849TCTGTATTTAGTCAAG 51 10806 10821 3240 561597 1836 1851 CCTCTGTATTTAGTCA72 10808 10823 3241 561598 1838 1853 GTCCTCTGTATTTAGT 55 10810 108253242 561599 1840 1855 CAGTCCTCTGTATTTA 63 10812 10827 3243 561600 18421857 ACCAGTCCTCTGTATT 66 10814 10829 3244 561601 1844 1859TTACCAGTCCTCTGTA 57 10816 10831 3245 561602 1846 1861 AATTACCAGTCCTCTG43 10818 10833 3246 561603 1848 1863 ACAATTACCAGTCCTC 67 10820 108353247

TABLE 149 Inhibition of ANGPTL3 mRNA by deoxy, MOE and (S)-cEt gapmerstargeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ ID SEQ ID ID NO: NO: ID NO: NO:2 SEQ 1 Start 1 Stop % 2 Start Stop ID ISIS NO Site Site Sequenceinhibition Site Site NO 561770 N/A N/A ACAAAGGTAGGTCACC 77 11576 11591143 586719 N/A N/A TCTGACAAGATTCCTT 76 11167 11182 3248 586720 N/A N/AATCTGACAAGATTCCT 79 11168 11183 3249 586721 N/A N/A TAATCTGACAAGATTC 5011170 11185 3250 586722 N/A N/A GTAATCTGACAAGATT 41 11171 11186 3251586723 N/A N/A CTTGTCAGGCTGTTTA 50 11312 11327 3252 586724 N/A N/AGCTTGTCAGGCTGTTT 81 11313 11328 3253 586725 N/A N/A ATGCTTGTCAGGCTGT 7811315 11330 3254 586726 N/A N/A TACATGCTTGTCAGGC 78 11318 11333 3255586727 N/A N/A ATACATGCTTGTCAGG 76 11319 11334 3256 586728 N/A N/AAAAGGTAGGTCACCAT 72 11574 11589 3257 586729 N/A N/A CAAAGGTAGGTCACCA 6911575 11590 3258 586730 N/A N/A GACAAAGGTAGGTCAC 55 11577 11592 3259586731 N/A N/A TGACAAAGGTAGGTCA 32 11578 11593 3260 586732 N/A N/ATCTGACATAGCTTTTT 63 5436 5451 3261 586733 N/A N/A ATTCTGACATAGCTTT 765438 5453 3262 586734 N/A N/A GATTCTGACATAGCTT 73 5439 5454 3263 586735N/A N/A GGATTCTGACATAGCT 81 5440 5455 3264 586736 N/A N/AATGGATTCTGACATAG 74 5442 5457 3265 586737 N/A N/A CATGGATTCTGACATA 725443 5458 3266 586738 N/A N/A ACATGGATTCTGACAT 59 5444 5459 3267 586739N/A N/A TACATGGATTCTGACA 71 5445 5460 3268 586740 N/A N/AATACATGGATTCTGAC 60 5446 5461 3269 586741 N/A N/A TTTAGCAGCACTACTA 655628 5643 3270 586742 N/A N/A TTTTAGCAGCACTACT 51 5629 5644 3271 586743N/A N/A CTTTTAGCAGCACTAC 74 5630 5645 3272 586744 N/A N/ACCTTTTAGCAGCACTA 83 5631 5646 223 586745 N/A N/A ACCTTTTAGCAGCACT 845632 5647 224 586746 N/A N/A AAACCTTTTAGCAGCA 87 5634 5649 225 586747N/A N/A AAAACCTTTTAGCAGC 80 5635 5650 3273 586748 N/A N/AGATAAAAAACCTTTTA 16 5640 5655 3274 586749 N/A N/A TGATAAAAAACCTTTT 255641 5656 3275 586750 N/A N/A AGATGTTGGCAGGTTG 72 6188 6203 3276 586751N/A N/A TAGATGTTGGCAGGTT 76 6189 6204 3277 586752 N/A N/AGTAGATGTTGGCAGGT 73 6190 6205 3278 586753 N/A N/A TGTAGATGTTGGCAGG 656191 6206 3279 586754 N/A N/A CTGTAGATGTTGGCAG 61 6192 6207 3280 586755N/A N/A ATCTGTAGATGTTGGC 84 6194 6209 226 586756 N/A N/ATATCTGTAGATGTTGG 71 6195 6210 3281 586757 N/A N/A ATATCTGTAGATGTTG 616196 6211 3282 586758 N/A N/A CATATCTGTAGATGTT 63 6197 6212 3283 586759N/A N/A TTTGAACCAGGCTTTC 47 6243 6258 3284 586760 N/A N/AAATTTGAACCAGGCTT 78 6245 6260 3285 586761 N/A N/A TAATTTGAACCAGGCT 836246 6261 227 586762 N/A N/A CATAATTTGAACCAGG 81 6248 6263 3286 586763N/A N/A ACATAATTTGAACCAG 36 6249 6264 3287 586764 N/A N/ATACATAATTTGAACCA 38 6250 6265 3288 586765 N/A N/A ATACATAATTTGAACC 156251 6266 3289 586766 N/A N/A ACATTGGTCGGAAAAC 43 6424 6439 3290 586767N/A N/A GACATTGGTCGGAAAA 49 6425 6440 3291 586768 N/A N/AAGACATTGGTCGGAAA 59 6426 6441 3292 586769 N/A N/A CAGACATTGGTCGGAA 666427 6442 3293 586770 N/A N/A GCAGACATTGGTCGGA 80 6428 6443 3294 586771N/A N/A AAGCAGACATTGGTCG 65 6430 6445 3295 586772 N/A N/ATGTACAGATTACCTGT 51 6506 6521 3296 586773 N/A N/A TTGTACAGATTACCTG 346507 6522 3297 586774 N/A N/A ATTGTACAGATTACCT 62 6508 6523 3298 586775N/A N/A GATTGTACAGATTACC 59 6509 6524 3299 586776 N/A N/AAGATTGTACAGATTAC 46 6510 6525 3300 586777 N/A N/A TCAGATTGTACAGATT 636512 6527 3301 586778 N/A N/A TTCAGATTGTACAGAT 63 6513 6528 3302 586779N/A N/A ATTCAGATTGTACAGA 71 6514 6529 3303 586780 N/A N/ATATTCAGATTGTACAG 55 6515 6530 3304 586781 N/A N/A TTATTCAGATTGTACA 526516 6531 3305 586782 N/A N/A TAGGTATGTCTTTTAT 52 6936 6951 3306 586783N/A N/A TGTCTTAGGTATGTCT 76 6941 6956 3307 586784 N/A N/AATTGTCTTAGGTATGT 73 6943 6958 3308 586785 N/A N/A GATTGTCTTAGGTATG 606944 6959 3309 586786 N/A N/A TTCTTAGATGGCGTGT 74 7207 7222 3310 586787N/A N/A TTTTCTTAGATGGCGT 86 7209 7224 228 586788 N/A N/AATTTTTCTTAGATGGC 75 7211 7226 3311 586789 N/A N/A CATTTTTCTTAGATGG 497212 7227 3312 586790 N/A N/A GCATTTTTCTTAGATG 47 7213 7228 3313 586791N/A N/A ATAAGTCCCAATTTTA 27 10066 10081 3314 586792 N/A N/ATATAAGTCCCAATTTT 27 10067 10082 3315 586793 N/A N/A GTATAAGTCCCAATTT 2810068 10083 3316 586794 N/A N/A TGTATAAGTCCCAATT 38 10069 10084 3317586795 N/A N/A CTGTATAAGTCCCAAT 69 10070 10085 3318 586796 N/A N/AATCTGTATAAGTCCCA 88 10072 10087 229 586797 N/A N/A AATCTGTATAAGTCCC 8410073 10088 230 586798 N/A N/A TAATCTGTATAAGTCC 58 10074 10089 3319586799 N/A N/A ATAATCTGTATAAGTC 21 10075 10090 3320 586800 N/A N/AAATAATCTGTATAAGT 12 10076 10091 3321 586801 N/A N/A TGCATGTATCCCAGTT 8010095 10110 3322 586802 N/A N/A ATGCATGTATCCCAGT 83 10096 10111 231586803 N/A N/A AGATGCATGTATCCCA 79 10098 10113 232 586804 N/A N/ATAGATGCATGTATCCC 87 10099 10114 3323 586805 N/A N/A TTAGATGCATGTATCC 7810100 10115 3324 586806 N/A N/A TTTAGATGCATGTATC 50 10101 10116 3325586653 7 22 GTGGAACTGTTTTCTT 63 3111 3126 3326 586656 9 24ACGTGGAACTGTTTTC 72 3113 3128 3327 586658 99 114 TTGATCAATTCTGGAG 743203 3218 3328 586660 101 116 TCTTGATCAATTCTGG 71 3205 3220 3329 561011102 117 GTCTTGATCAATTCTG 91 3206 3221 114 586661 103 118TGTCTTGATCAATTCT 85 3207 3222 209 586663 134 149 GGCTCTGGAGATAGAG 633238 3253 3330 586665 136 151 TTGGCTCTGGAGATAG 63 3240 3255 3331 586668140 155 GATTTTGGCTCTGGAG 64 3244 3259 3332 586669 142 157TTGATTTTGGCTCTGG 89 3246 3261 210 561026 143 158 CTTGATTTTGGCTCTG 843247 3262 117 586670 144 159 TCTTGATTTTGGCTCT 71 3248 3263 3333 586671146 161 AATCTTGATTTTGGCT 70 3250 3265 3334 586672 148 163CAAATCTTGATTTTGG 81 3252 3267 3335 586673 298 313 GCAGCGATAGATCATA 763402 3417 3336 586674 300 315 TTGCAGCGATAGATCA 76 3404 3419 3337 586675304 319 TGGTTTGCAGCGATAG 82 3408 3423 3338 586676 306 321ACTGGTTTGCAGCGAT 89 3410 3425 211 586677 315 330 TTTGATTTCACTGGTT 623419 3434 3339 586678 317 332 TCTTTGATTTCACTGG 66 3421 3436 3340 586679342 357 AGTTCTTCTCAGTTCC 77 3446 3461 3341 586680 476 491TTAGTTAGTTGCTCTT 65 3580 3595 3342 586681 478 493 AGTTAGTTAGTTGCTC 693582 3597 3343 586682 703 718 GTGCTCTTGGCTTGGA 78 6716 6731 3344 586683705 720 TGGTGCTCTTGGCTTG 77 6718 6733 3345 586684 802 817TATGTTCACCTCTGTT 55 7387 7402 3346 586685 804 819 TGTATGTTCACCTCTG 797389 7404 3347 586686 1260 1275 ACACTCATCATGCCAC 72 10232 10247 3348586687 1262 1277 CCACACTCATCATGCC 82 10234 10249 3349 586688 1308 1323AGATTTTGCTCTTGGT 87 10280 10295 212 586689 1310 1325 TTAGATTTTGCTCTTG 7810282 10297 3350 586690 1351 1366 CATTTTGAGACTTCCA 91 10323 10338 213586691 1353 1368 TCCATTTTGAGACTTC 86 10325 10340 214 586692 1365 1380AGAGTATAACCTTCCA 88 10337 10352 220 586693 1367 1382 ATAGAGTATAACCTTC 6910339 10354 3351 586694 1402 1417 AATCTGTTGGATGGAT 59 10374 10389 3352586695 1404 1419 TGAATCTGTTGGATGG 79 10376 10391 3353 586696 1420 1435TTCATTCAAAGCTTTC 82 10392 10407 3354 586697 1422 1437 AGTTCATTCAAAGCTT73 10394 10409 3355 561463 1423 1438 CAGTTCATTCAAAGCT 88 10395 10410 127586698 1424 1439 TCAGTTCATTCAAAGC 69 10396 10411 3356 586699 1488 1503GATTATTAGACCACAT 63 10460 10475 3357 586700 1490 1505 CAGATTATTAGACCAC90 10462 10477 221 561487 1491 1506 CCAGATTATTAGACCA 95 10463 10478 131586701 1492 1507 ACCAGATTATTAGACC 85 10464 10479 215 586702 1552 1567TAGACAGTGACTTTAA 83 10524 10539 216 586703 1554 1569 AATAGACAGTGACTTT 7010526 10541 3358 586704 1605 1620 AGAAATGTAAACGGTA 76 10577 10592 3359586705 1607 1622 TGAGAAATGTAAACGG 83 10579 10594 217 586706 1762 1777TCATATGATGCCTTTT 69 10734 10749 3360 586707 1764 1779 GCTCATATGATGCCTT84 10736 10751 218 586708 1766 1781 TAGCTCATATGATGCC 83 10738 10753 222561567 1767 1782 TTAGCTCATATGATGC 81 10739 10754 177 586709 1768 1783ATTAGCTCATATGATG 40 10740 10755 3361 586710 1774 1789 TGTGATATTAGCTCAT73 10746 10761 3362 586711 1776 1791 GTTGTGATATTAGCTC 80 10748 107633363 586712 1905 1920 TACTCTGTGCTGACGA 81 10877 10892 3364 586713 19071922 CATACTCTGTGCTGAC 81 10879 10894 3365 586714 2052 2067GTTTAAAGACAGCGAA 72 11024 11039 3366 586715 2054 2069 TTGTTTAAAGACAGCG81 11026 11041 3367 586716 2068 2083 GTAGTCATCTCCATTT 63 11040 110553368 586717 2070 2085 TAGTAGTCATCTCCAT 74 11042 11057 3369 561650 20712086 TTAGTAGTCATCTCCA 79 11043 11058 142 586718 2072 2087CTTAGTAGTCATCTCC 84 11044 11059 219

Example 120: Antisense Inhibition of Human ANGPTL3 in Hep3B Cells byDeoxy, MOE and (S)-cEt Gapmers

Additional antisense oligonucleotides were designed targeting an ANGPTL3nucleic acid and were tested for their effects on ANGPTL3 mRNA in vitro.ISIS 337487 and ISIS 233717, which are 5-10-5 MOE gapmers, were alsoincluded in the assay as benchmark oligonucleotides. Cultured Hep3Bcells at a density of 20,000 cells per well were transfected usingelectroporation with 4,500 nM antisense oligonucleotide. After atreatment period of approximately 24 hours, RNA was isolated from thecells and ANGPTL3 mRNA levels were measured by quantitative real-timePCR. Human primer probe set RTS3492_MGB was used to measure mRNA levels.ANGPTL3 mRNA levels were adjusted according to total RNA content, asmeasured by RIBOGREEN®. Results are presented as percent inhibition ofANGPTL3, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in the Tablesbelow were designed as deoxy, MOE, and (S)-cEt oligonucleotides or5-10-5 MOE gapmers. The deoxy, MOE and (S)-cEt oligonucleotides are 16nucleosides in length wherein the nucleoside have either a MOE sugarmodification, an (S)-cEt sugar modification, or a deoxy sugar residue.The sugar modifications of each antisense oligonucleotide is describedas ‘eek-d10-kke’, where ‘k’ indicates an (S)-cEt sugar modification; ‘d’indicates deoxyribose; the number indicates the number ofdeoxyribosesugars residues; and ‘e’ indicates a MOE modification. The 5-10-5 MOEgapmers are 20 nucleosides in length, wherein the central gap segmentcomprises often 2′-deoxynucleosides and is flanked by wing segments onthe 5′ direction and the 3′ direction comprising five nucleosides each.The internucleoside linkages throughout each oligonucleotide arephosphorothioate (P═S) linkages. All cytosine residues throughout eacholigonucleotide are 5-methylcytosines. “Start site” indicates the5′-most nucleoside to which the oligonucleotide is targeted in the humangene sequence. “Stop site” indicates the 3′-most nucleoside to which theoligonucleotide is targeted human gene sequence. Each oligonucleotidelisted in the Tables below is targeted to either the human ANGPTL3 mRNA,designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_014495.2) orthe human ANGPTL3 genomic sequence, designated herein as SEQ ID NO: 2(GENBANK Accession No. NT_032977.9 truncated from nucleotides 33032001to 33046000). ‘n/a’ indicates that the antisense oligonucleotide doesnot target that particular gene sequence with 100% complementarity.

TABLE 150 Inhibition of ANGPTL3 mRNA by oligonucleotides targeting SEQID NO: 1 and 2 SEQ SEQ SEQ ID SEQ ID ID ID NO: 1 NO: NO: 2 NO: 2 Start 1Stop % Start Stop SEQ ID ISIS NO Site Site Sequence Chemistry inhibitionSite Site NO 561671 N/A N/A TCTTAACTCTATATAT Deoxy, MOE, and cEt 12 30763091 3370 561672 N/A N/A CTTCTTAACTCTATAT Deoxy, MOE, and cEt 12 30783093 3371 561673 N/A N/A GACTTCTTAACTCTAT Deoxy, MOE, and cEt 18 30803095 3372 561674 N/A N/A TAGACTTCTTAACTCT Deoxy, MOE, and cEt 20 30823097 3373 561675 N/A N/A CCTAGACTTCTTAACT Deoxy, MOE, and cEt 9 30843099 3374 561676 N/A N/A GACCTAGACTTCTTAA Deoxy, MOE, and cEt 0 30863101 3375 561677 N/A N/A CAGACCTAGACTTCTT Deoxy, MOE, and cEt 18 30883103 3376 561678 N/A N/A AGCAGACCTAGACTTC Deoxy, MOE, and cEt 26 30903105 3377 561679 N/A N/A GAAGCAGACCTAGACT Deoxy, MOE, and cEt 24 30923107 3378 561680 N/A N/A TGGAAGCAGACCTAGA Deoxy, MOE, and cEt 30 30943109 3379 561758 N/A N/A CTTTAACAAATGGGTT Deoxy, MOE, and cEt 25 1153911554 3380 561759 N/A N/A ATCCTTTAACAAATGG Deoxy, MOE, and cEt 31 1154211557 3381 561760 N/A N/A CTATATCCTTTAACAA Deoxy, MOE, and cEt 28 1154611561 3382 561761 N/A N/A GCACTATATCCTTTAA Deoxy, MOE, and cEt 59 1154911564 3383 561762 N/A N/A TGGGCACTATATCCTT Deoxy, MOE, and cEt 34 1155211567 3384 561763 N/A N/A ACTTGGGCACTATATC Deoxy, MOE, and cEt 30 1155511570 3385 561764 N/A N/A ATAACTTGGGCACTAT Deoxy, MOE, and cEt 51 1155811573 3386 561765 N/A N/A CATATAACTTGGGCAC Deoxy, MOE, and cEt 47 1156111576 3387 561766 N/A N/A CACCATATAACTTGGG Deoxy, MOE, and cEt 47 1156411579 3388 561767 N/A N/A GGTCACCATATAACTT Deoxy, MOE, and cEt 58 1156711582 3389 561768 N/A N/A GTAGGTCACCATATAA Deoxy, MOE, and cEt 62 1157011585 3390 561769 N/A N/A AAGGTAGGTCACCATA Deoxy, MOE, and cEt 65 1157311588 3391 561770 N/A N/A ACAAAGGTAGGTCACC Deoxy, MOE, and cEt 73 1157611591 143 561771 N/A N/A TTGACAAAGGTAGGTC Deoxy, MOE, and cEt 70 1157911594 3392 561772 N/A N/A GTATTGACAAAGGTAG Deoxy, MOE, and cEt 58 1158211597 3393 561773 N/A N/A TAAGTATTGACAAAGG Deoxy, MOE, and cEt 42 1158511600 3394 561774 N/A N/A TGCTAAGTATTGACAA Deoxy, MOE, and cEt 51 1158811603 3395 561775 N/A N/A TAATGCTAAGTATTGA Deoxy, MOE, and cEt 42 1159111606 3396 561776 N/A N/A TACATAATGCTAAGTA Deoxy, MOE, and cEt 36 1159511610 3397 561777 N/A N/A GGATAATTTGAAATAC Deoxy, MOE, and cEt 24 1160811623 3398 561778 N/A N/A TATTGGATAATTTGAA Deoxy, MOE, and cEt 35 1161211627 3399 561779 N/A N/A GTATATTGGATAATTT Deoxy, MOE, and cEt 0 1161511630 3400 561780 N/A N/A CATGTATATTGGATAA Deoxy, MOE, and cEt 20 1161811633 3401 561781 N/A N/A TGACATGTATATTGGA Deoxy, MOE, and cEt 73 1162111636 144 561782 N/A N/A CTTTTATATATGTGAC Deoxy, MOE, and cEt 37 1165211667 3402 561783 N/A N/A GATCATACATATCTTT Deoxy, MOE, and cEt 51 1166411679 3403 561784 N/A N/A ATAGATCATACATATC Deoxy, MOE, and cEt 46 1166711682 3404 561785 N/A N/A CACATAGATCATACAT Deoxy, MOE, and cEt 65 1167011685 3405 561786 N/A N/A ATTCACATAGATCATA Deoxy, MOE, and cEt 48 1167311688 3406 561787 N/A N/A AGGATTCACATAGATC Deoxy, MOE, and cEt 48 1167611691 3407 561788 N/A N/A CTTAGGATTCACATAG Deoxy, MOE, and cEt 42 1167911694 3408 561789 N/A N/A TTACTTAGGATTCACA Deoxy, MOE, and cEt 58 1168211697 3409 561790 N/A N/A TATTTACTTAGGATTC Deoxy, MOE, and cEt 45 1168511700 3410 561791 N/A N/A GTACTTTTCTGGAACA Deoxy, MOE, and cEt 77 1170411719 145 561792 N/A N/A CCTGAAAATTATAGAT Deoxy, MOE, and cEt 35 1174111756 3411 561793 N/A N/A GGTCCTGAAAATTATA Deoxy, MOE, and cEt 32 1174411759 3412 561794 N/A N/A TGTGGTCCTGAAAATT Deoxy, MOE, and cEt 45 1174711762 3413 561795 N/A N/A GTCTGTGGTCCTGAAA Deoxy, MOE, and cEt 47 1175011765 3414 561796 N/A N/A TTAGTCTGTGGTCCTG Deoxy, MOE, and cEt 67 1175311768 3415 561797 N/A N/A AGCTTAGTCTGTGGTC Deoxy, MOE, and cEt 55 1175611771 3416 561798 N/A N/A GACAGCTTAGTCTGTG Deoxy, MOE, and cEt 47 1175911774 3417 561799 N/A N/A TTCGACAGCTTAGTCT Deoxy, MOE, and cEt 68 1176211777 3418 561800 N/A N/A AATTTCGACAGCTTAG Deoxy, MOE, and cEt 61 1176511780 3419 561801 N/A N/A GTTAATTTCGACAGCT Deoxy, MOE, and cEt 70 1176811783 3420 561802 N/A N/A CCTAAAAAAATCAGCG Deoxy, MOE, and cEt 19 1178311798 3421 561803 N/A N/A GGCCCTAAAAAAATCA Deoxy, MOE, and cEt 0 1178611801 3422 561804 N/A N/A TTCTGGCCCTAAAAAA Deoxy, MOE, and cEt 10 1179011805 3423 561805 N/A N/A GTATTCTGGCCCTAAA Deoxy, MOE, and cEt 44 1179311808 3424 561806 N/A N/A TTGGTATTCTGGCCCT Deoxy, MOE, and cEt 45 1179611811 3425 561807 N/A N/A ATTTTGGTATTCTGGC Deoxy, MOE, and cEt 59 1179911814 3426 561808 N/A N/A GCCATTTTGGTATTCT Deoxy, MOE, and cEt 58 1180211817 3427 561809 N/A N/A GGAGCCATTTTGGTAT Deoxy, MOE, and cEt 33 1180511820 3428 561810 N/A N/A AGAGGAGCCATTTTGG Deoxy, MOE, and cEt 36 1180811823 3429 561811 N/A N/A AAGAGAGGAGCCATTT Deoxy, MOE, and cEt 14 1181111826 3430 561812 N/A N/A ATTGTCCAATTTTGGG Deoxy, MOE, and cEt 25 1182911844 3431 561813 N/A N/A GAAATTGTCCAATTTT Deoxy, MOE, and cEt 38 1183211847 3432 561814 N/A N/A TTTGAAATTGTCCAAT Deoxy, MOE, and cEt 36 1183511850 3433 561815 N/A N/A GCATTTGAAATTGTCC Deoxy, MOE, and cEt 67 1183811853 3434 561816 N/A N/A GCAACTCATATATTAA Deoxy, MOE, and cEt 57 1186911884 3435 561817 N/A N/A GAAGCAACTCATATAT Deoxy, MOE, and cEt 46 1187211887 3436 561818 N/A N/A GAGGAAGCAACTCATA Deoxy, MOE, and cEt 14 1187511890 3437 561819 N/A N/A ATAGAGGAAGCAACTC Deoxy, MOE, and cEt 60 1187811893 3438 561820 N/A N/A CAAATAGAGGAAGCAA Deoxy, MOE, and cEt 36 1188111896 3439 561821 N/A N/A AACCAAATAGAGGAAG Deoxy, MOE, and cEt 38 1188411899 3440 561822 N/A N/A GGAAACCAAATAGAGG Deoxy, MOE, and cEt 51 1188711902 3441 561823 N/A N/A CTTTAAGTGAAGTTAC Deoxy, MOE, and cEt 30 36363651 3442 561824 N/A N/A TACTTACTTTAAGTGA Deoxy, MOE, and cEt 27 36423657 3443 561825 N/A N/A GAACCCTCTTTATTTT Deoxy, MOE, and cEt 25 36593674 3444 561826 N/A N/A AAACATGAACCCTCTT Deoxy, MOE, and cEt 14 36653680 3445 561827 N/A N/A GATCCACATTGAAAAC Deoxy, MOE, and cEt 0 36833698 3446 561828 N/A N/A CATGCCTTAGAAATAT Deoxy, MOE, and cEt 33 37103725 3447 561829 N/A N/A AAATGGCATGCCTTAG Deoxy, MOE, and cEt 46 37163731 3448 561830 N/A N/A GTATTTCAAATGGCAT Deoxy, MOE, and cEt 54 37233738 3449 561831 N/A N/A GCAACAAAGTATTTCA Deoxy, MOE, and cEt 60 37313746 3450 561832 N/A N/A GTATTTCAACAATGCA Deoxy, MOE, and cEt 28 37443759 3451 561833 N/A N/A ATAACATTAGGGAAAC Deoxy, MOE, and cEt 18 38273842 3452 561834 N/A N/A TCATATATAACATTAG Deoxy, MOE, and cEt 18 38333848 3453 561912 N/A N/A GTGGTTTTGAGCAAAG Deoxy, MOE, and cEt 5 47364751 3454 561913 N/A N/A CTATTGTGTGGTTTTG Deoxy, MOE, and cEt 36 47434758 3455 561914 N/A N/A GGAAAGCTATTGTGTG Deoxy, MOE, and cEt 18 47494764 3456 561915 N/A N/A TATGAGTGAAATGGAA Deoxy, MOE, and cEt 13 47614776 3457 561916 N/A N/A AGCCAATATGAGTGAA Deoxy, MOE, and cEt 57 47674782 3458 561917 N/A N/A CTAAAGAGCCAATATG Deoxy, MOE, and cEt 33 47734788 3459 561918 N/A N/A CTTGGTCTAAAGAGCC Deoxy, MOE, and cEt 70 47794794 146 561919 N/A N/A GGTAATCTTGGTCTAA Deoxy, MOE, and cEt 46 47854800 3460 561920 N/A N/A GATGACGAAGGGTTGG Deoxy, MOE, and cEt 28 48004815 3461 561921 N/A N/A CAGTGAGATGACGAAG Deoxy, MOE, and cEt 39 48064821 3462 561922 N/A N/A TGAAGTCAGTGAGATG Deoxy, MOE, and cEt 49 48124827 3463 561923 N/A N/A AGGAGGTGAAGTCAGT Deoxy, MOE, and cEt 35 48184833 3464 561924 N/A N/A GAGTAGAGGAGGTGAA Deoxy, MOE, and cEt 33 48244839 3465 561925 N/A N/A TAACTAGAGTAGAGGA Deoxy, MOE, and cEt 35 48304845 3466 561926 N/A N/A TCAGAATAACTAGAGT Deoxy, MOE, and cEt 24 48364851 3467 561927 N/A N/A AAGCGGTCAGAATAAC Deoxy, MOE, and cEt 39 48424857 3468 561928 N/A N/A CTGGTAAAGCGGTCAG Deoxy, MOE, and cEt 51 48484863 3469 561929 N/A N/A TGAATACTGGTAAAGC Deoxy, MOE, and cEt 63 48544869 3470 561930 N/A N/A TGTGTTTGAATACTGG Deoxy, MOE, and cEt 65 48604875 3471 561931 N/A N/A GTTTGATGTGTTTGAA Deoxy, MOE, and cEt 49 48664881 3472 561932 N/A N/A CAGTATGTTTGATGTG Deoxy, MOE, and cEt 48 48724887 3473 561933 N/A N/A AGGTGGCAGTATGTTT Deoxy, MOE, and cEt 0 48784893 3474 561934 N/A N/A GCTTTGAGGTGGCAGT Deoxy, MOE, and cEt 48 48844899 3475 561935 N/A N/A GGGCAAAGGCTTTGAG Deoxy, MOE, and cEt 28 48924907 3476 561936 N/A N/A CAACAAGGGCAAAGGC Deoxy, MOE, and cEt 65 48984913 3477 561937 N/A N/A GAGGAAACAACAAGGG Deoxy, MOE, and cEt 42 49054920 3478 561938 N/A N/A CCAGTTAGAGGAAACA Deoxy, MOE, and cEt 52 49124927 3479 561939 N/A N/A CCAGGGCAGAAGAGCG Deoxy, MOE, and cEt 61 49304945 3480 561940 N/A N/A TAGATACCAGGGCAGA Deoxy, MOE, and cEt 68 49364951 3481 561941 N/A N/A CAGAGAGTGGGCCACG Deoxy, MOE, and cEt 46 49524967 3482 561942 N/A N/A GGAAATCAGAGAGTGG Deoxy, MOE, and cEt 42 49584973 3483 561943 N/A N/A CCTAAGGGAAATCAGA Deoxy, MOE, and cEt 26 49644979 3484 561944 N/A N/A AACGACCCTAAGGGAA Deoxy, MOE, and cEt 45 49704985 3485 561945 N/A N/A TTTGATAACGACCCTA Deoxy, MOE, and cEt 57 49764991 3486 561946 N/A N/A TTTTTGTTTGATAACG Deoxy, MOE, and cEt 21 49824997 3487 561947 N/A N/A CATTGGGAATTTTTTG Deoxy, MOE, and cEt 35 49925007 3488 561948 N/A N/A AGTCTTCATTGGGAAT Deoxy, MOE, and cEt 69 49985013 3489 561949 N/A N/A CTTGTAAGTCTTCATT Deoxy, MOE, and cEt 35 50045019 3490 561950 N/A N/A AGTGACCTTGTAAGTC Deoxy, MOE, and cEt 56 50105025 3491 561951 N/A N/A TGGTTAAGTGACCTTG Deoxy, MOE, and cEt 67 50165031 3492 561952 N/A N/A GATTTTTGGTTAAGTG Deoxy, MOE, and cEt 43 50225037 3493 561953 N/A N/A GGTTGTGATTTTTGGT Deoxy, MOE, and cEt 58 50285043 3494 561954 N/A N/A CCAGGCGGTTGTGATT Deoxy, MOE, and cEt 49 50345049 3495 561955 N/A N/A ATGGGACCAGGCGGTT Deoxy, MOE, and cEt 52 50405055 3496 561956 N/A N/A AAGTTTTCAGGGATGG Deoxy, MOE, and cEt 49 50525067 3497 561957 N/A N/A AAGTAGAAGTTTTCAG Deoxy, MOE, and cEt 16 50585073 3498 561958 N/A N/A CTAAGGAAGTAGAAGT Deoxy, MOE, and cEt 33 50645079 3499 561959 N/A N/A AAGTAGCTAAGGAAGT Deoxy, MOE, and cEt 35 50705085 3500 561960 N/A N/A GGAGAAAAGTAGCTAA Deoxy, MOE, and cEt 36 50765091 3501 561961 N/A N/A TGTGCAGGAGAAAAGT Deoxy, MOE, and cEt 53 50825097 3502 561962 N/A N/A GGTGAGTGTGCAGGAG Deoxy, MOE, and cEt 44 50885103 3503 561963 N/A N/A AATAAAGGTGAGTGTG Deoxy, MOE, and cEt 38 50945109 3504 561964 N/A N/A TGCAGGAATAGAAGAG Deoxy, MOE, and cEt 58 51385153 3505 561965 N/A N/A TTTTAGTGCAGGAATA Deoxy, MOE, and cEt 20 51445159 3506 561966 N/A N/A TATTCACAGAGCTTAC Deoxy, MOE, and cEt 63 51615176 3507 561967 N/A N/A TCCCTGTATTCACAGA Deoxy, MOE, and cEt 61 51675182 3508 561968 N/A N/A GAAAAAATCCCTGTAT Deoxy, MOE, and cEt 22 51745189 3509 561969 N/A N/A TATGAAGATAATGGAA Deoxy, MOE, and cEt 34 51875202 3510 561970 N/A N/A GGAGTATATACAAATA Deoxy, MOE, and cEt 46 52115226 3511 561971 N/A N/A TATTCTGGAGTATATA Deoxy, MOE, and cEt 29 52175232 3512 561972 N/A N/A ATTCTATATTCTGGAG Deoxy, MOE, and cEt 58 52235238 3513 561973 N/A N/A CATACAGTATTCTATA Deoxy, MOE, and cEt 39 52315246 3514 561974 N/A N/A GTGTGCCATACAGTAT Deoxy, MOE, and cEt 48 52375252 3515 561975 N/A N/A AGAAATGCCTACTGTG Deoxy, MOE, and cEt 34 52505265 3516 561976 N/A N/A ATTCAACAGAAATGCC Deoxy, MOE, and cEt 52 52575272 3517 561977 N/A N/A GAATATGACATTACAT Deoxy, MOE, and cEt 33 52795294 3518 561978 N/A N/A CTGTGTGAATATGACA Deoxy, MOE, and cEt 63 52855300 3519 561979 N/A N/A ACGCTTCTGTGTGAAT Deoxy, MOE, and cEt 59 52915306 3520 561980 N/A N/A TAGCACACGCTTCTGT Deoxy, MOE, and cEt 29 52975312 3521 561981 N/A N/A TAATCATAGCACACGC Deoxy, MOE, and cEt 64 53035318 3522 561982 N/A N/A CCAAGTAATAATAATC Deoxy, MOE, and cEt 26 53145329 3523 561983 N/A N/A AGTAATCCAAGTAATA Deoxy, MOE, and cEt 33 53205335 3524 561984 N/A N/A ATTTCTAGTAATCCAA Deoxy, MOE, and cEt 42 53265341 3525 561985 N/A N/A CACACTATTTCTAGTA Deoxy, MOE, and cEt 40 53325347 3526 561986 N/A N/A ATGAGGCACACTATTT Deoxy, MOE, and cEt 47 53385353 3527 561987 N/A N/A TTAATTATGAGGCACA Deoxy, MOE, and cEt 58 53445359 3528 561988 N/A N/A TGACCTTTAATTATGA Deoxy, MOE, and cEt 38 53505365 3529 562066 N/A N/A GCAATTTATTGAATGA Deoxy, MOE, and cEt 27 60836098 3530 562067 N/A N/A GGGTTTGCAATTTATT Deoxy, MOE, and cEt 38 60896104 3531 562068 N/A N/A TGTGTTGGGTTTGCAA Deoxy, MOE, and cEt 43 60956110 3532 562069 N/A N/A TTTAAGTGTGTTGGGT Deoxy, MOE, and cEt 71 61016116 3533 562070 N/A N/A GTTTAGCAGTAACATT Deoxy, MOE, and cEt 38 61266141 3534 562071 N/A N/A ATTCAGTAGTTTATCG Deoxy, MOE, and cEt 17 61456160 3535 562072 N/A N/A CTATATATTCAGTAGT Deoxy, MOE, and cEt 0 61516166 3536 562073 N/A N/A GCTTACTTTCTATATA Deoxy, MOE, and cEt 21 61606175 3537 562074 N/A N/A AGTTTGTTTGCTTACT Deoxy, MOE, and cEt 63 61696184 3538 562075 N/A N/A TTGGCAAGTTTGTTTG Deoxy, MOE, and cEt 55 61756190 3539 562076 N/A N/A GGCAGGTTGGCAAGTT Deoxy, MOE, and cEt 68 61816196 3540 562077 N/A N/A GATGTTGGCAGGTTGG Deoxy, MOE, and cEt 54 61876202 3541 562078 N/A N/A TCTGTAGATGTTGGCA Deoxy, MOE, and cEt 81 61936208 147 562079 N/A N/A AACATATCTGTAGATG Deoxy, MOE, and cEt 32 61996214 3542 562080 N/A N/A CCTGTAAACATATCTG Deoxy, MOE, and cEt 51 62056220 3543 562081 N/A N/A TTTTGACCTGTAAACA Deoxy, MOE, and cEt 14 62116226 3544 562082 N/A N/A GATAATTTTTGACCTG Deoxy, MOE, and cEt 49 62176232 3545 562083 N/A N/A TCTTGATAATTTGATA Deoxy, MOE, and cEt 13 62296244 3546 562084 N/A N/A AGGCTTTCTTGATAAT Deoxy, MOE, and cEt 55 62356250 3547 562085 N/A N/A TGAACCAGGCTTTCTT Deoxy, MOE, and cEt 74 62416256 3548 562086 N/A N/A ATAATTTGAACCAGGC Deoxy, MOE, and cEt 82 62476262 148 562087 N/A N/A GATAAAGACATAATAC Deoxy, MOE, and cEt 21 62636278 3549 562088 N/A N/A ACCTGTGATAAAGACA Deoxy, MOE, and cEt 27 62696284 3550 562089 N/A N/A CTTCAGACCTGTGATA Deoxy, MOE, and cEt 23 62756290 3551 562090 N/A N/A ACTGATCTTCAGACCT Deoxy, MOE, and cEt 48 62816296 3552 562091 N/A N/A GGTCTTACTGATCTTC Deoxy, MOE, and cEt 59 62876302 3553 562092 N/A N/A GTTTTAGGTCTTACTG Deoxy, MOE, and cEt 21 62936308 3554 562093 N/A N/A GTTCAGATTTTAAGTT Deoxy, MOE, and cEt 31 63216336 3555 562094 N/A N/A ATATTCTGTTCAGATT Deoxy, MOE, and cEt 36 63286343 3556 562095 N/A N/A ATATTGTAATGTATTC Deoxy, MOE, and cEt 52 63726387 3557 562096 N/A N/A CTTAGAATATTGTAAT Deoxy, MOE, and cEt 13 63786393 3558 562097 N/A N/A GCTTTGCTTAGAATAT Deoxy, MOE, and cEt 47 63846399 3559 562098 N/A N/A GAGACTGCTTTGCTTA Deoxy, MOE, and cEt 48 63906405 3560 562099 N/A N/A AAAGTAGAGACTGCTT Deoxy, MOE, and cEt 44 63966411 3561 562100 N/A N/A AGGCCAAAAGTAGAGA Deoxy, MOE, and cEt 59 64026417 3562 562101 N/A N/A TCGGAAAACAGAGCAA Deoxy, MOE, and cEt 63 64176432 3563 562102 N/A N/A CATTGGTCGGAAAACA Deoxy, MOE, and cEt 53 64236438 3564 562103 N/A N/A AGCAGACATTGGTCGG Deoxy, MOE, and cEt 83 64296444 149 562104 N/A N/A AGCAAGGCAAAAAAGC Deoxy, MOE, and cEt 22 64426457 3565 562105 N/A N/A GACATTATTTAATAAG Deoxy, MOE, and cEt 21 64706485 3566 562106 N/A N/A ATCAGGGACATTATTT Deoxy, MOE, and cEt 34 64766491 3567 562107 N/A N/A TATTTAATCAGGGACA Deoxy, MOE, and cEt 47 64826497 3568 562108 N/A N/A ATTACCTGTTCTCAAA Deoxy, MOE, and cEt 30 64996514 3569 562109 N/A N/A GTACAGATTACCTGTT Deoxy, MOE, and cEt 38 65056520 3570 562110 N/A N/A CAGATTGTACAGATTA Deoxy, MOE, and cEt 76 65116526 150 562111 N/A N/A GTTATTCAGATTGTAC Deoxy, MOE, and cEt 32 65176532 3571 562112 N/A N/A AACAGTGTTATTCAGA Deoxy, MOE, and cEt 58 65236538 3572 562113 N/A N/A TAGATAAACAGTGTTA Deoxy, MOE, and cEt 33 65296544 3573 562114 N/A N/A TGATATTTAGATAAAC Deoxy, MOE, and cEt 26 65366551 3574 562115 N/A N/A GGTGTTTGATATTTAG Deoxy, MOE, and cEt 60 65426557 3575 562116 N/A N/A TATAACGGTGTTTGAT Deoxy, MOE, and cEt 42 65486563 3576 562117 N/A N/A TAATGTTATAACGGTG Deoxy, MOE, and cEt 62 65546569 3577 562118 N/A N/A AGTTCATAATGTTATA Deoxy, MOE, and cEt 21 65606575 3578 562119 N/A N/A GTCTTTCAGTTCATAA Deoxy, MOE, and cEt 57 65676582 3579 562120 N/A N/A ACAGTTTGTCTTTCAG Deoxy, MOE, and cEt 59 65746589 3580 562121 N/A N/A AGAAGTACAGTTTGTC Deoxy, MOE, and cEt 3 65806595 3581 562122 N/A N/A GATGTCAGAAGTACAG Deoxy, MOE, and cEt 45 65866601 3582 562123 N/A N/A AGTAAGGATGTCAGAA Deoxy, MOE, and cEt 44 65926607 3583 562124 N/A N/A AATCTGAGTAAGGATG Deoxy, MOE, and cEt 45 65986613 3584 562125 N/A N/A GAATATACAATTAGGG Deoxy, MOE, and cEt 13 66166631 3585 562126 N/A N/A TGATACTGAATATACA Deoxy, MOE, and cEt 13 66236638 3586 562127 N/A N/A CTGAGCTGATAAAAGA Deoxy, MOE, and cEt 1 66606675 3587 562128 N/A N/A ACCATCATGTTTTACA Deoxy, MOE, and cEt 44 67726787 3588 562129 N/A N/A TGTCTTACCATCATGT Deoxy, MOE, and cEt 29 67786793 3589 562130 N/A N/A CCAAAGTGTCTTACCA Deoxy, MOE, and cEt 42 67846799 3590 562131 N/A N/A AACCCACCAAAGTGTC Deoxy, MOE, and cEt 33 67906805 3591 562132 N/A N/A GAAGGAAACCCACCAA Deoxy, MOE, and cEt 24 67966811 3592 562133 N/A N/A CTTCAAGAAGGAAACC Deoxy, MOE, and cEt 28 68026817 3593 562134 N/A N/A TAATAGCTTCAAGAAG Deoxy, MOE, and cEt 1 68086823 3594 562135 N/A N/A GGGAATTTGATAATAA Deoxy, MOE, and cEt 0 68216836 3595 562136 N/A N/A AGAATAGGGAATTTGA Deoxy, MOE, and cEt 18 68276842 3596 562137 N/A N/A GTCCTAAGAATAGGGA Deoxy, MOE, and cEt 9 68336848 3597 562138 N/A N/A GAACAAGTCCTAAGAA Deoxy, MOE, and cEt 7 68396854 3598 562139 N/A N/A AGTCTAGAACAAGTCC Deoxy, MOE, and cEt 70 68456860 3599 562140 N/A N/A TCTTTTAGTCTAGAAC Deoxy, MOE, and cEt 22 68516866 3600 562141 N/A N/A TAACTATCTTTTAGTC Deoxy, MOE, and cEt 15 68576872 3601 562142 N/A N/A ATCTCTTAACTATCTT Deoxy, MOE, and cEt 35 68636878 3602 560991 3 18 AACTGTTTTCTTCTGG Deoxy, MOE, and cEt 37 3107 31223603 560992 8 23 CGTGGAACTGTTTTCT Deoxy, MOE, and cEt 74 3112 3127 112560993 22 37 TCAATTTCAAGCAACG Deoxy, MOE, and cEt 68 3126 3141 3604560994 51 66 CTTAATTGTGAACATT Deoxy, MOE, and cEt 21 3155 3170 3605560995 53 68 AGCTTAATTGTGAACA Deoxy, MOE, and cEt 59 3157 3172 3606560996 55 70 GGAGCTTAATTGTGAA Deoxy, MOE, and cEt 0 3159 3174 3607560997 57 72 AAGGAGCTTAATTGTG Deoxy, MOE, and cEt 36 3161 3176 3608560998 59 74 AGAAGGAGCTTAATTG Deoxy, MOE, and cEt 47 3163 3178 3609560999 61 76 AAAGAAGGAGCTTAAT Deoxy, MOE, and cEt 20 3165 3180 3610561000 76 91 CTAGAGGAACAATAAA Deoxy, MOE, and cEt 23 3180 3195 3611561001 79 94 TAACTAGAGGAACAAT Deoxy, MOE, and cEt 19 3183 3198 3612561002 81 96 AATAACTAGAGGAACA Deoxy, MOE, and cEt 38 3185 3200 3613561003 84 99 GGAAATAACTAGAGGA Deoxy, MOE, and cEt 48 3188 3203 3614561004 86 101 GAGGAAATAACTAGAG Deoxy, MOE, and cEt 37 3190 3205 3615561005 88 103 TGGAGGAAATAACTAG Deoxy, MOE, and cEt 68 3192 3207 3616561006 90 105 TCTGGAGGAAATAACT Deoxy, MOE, and cEt 49 3194 3209 3617561007 94 109 CAATTCTGGAGGAAAT Deoxy, MOE, and cEt 43 3198 3213 3618561008 96 111 ATCAATTCTGGAGGAA Deoxy, MOE, and cEt 73 3200 3215 3619561009 98 113 TGATCAATTCTGGAGG Deoxy, MOE, and cEt 72 3202 3217 3620561010 100 115 CTTGATCAATTCTGGA Deoxy, MOE, and cEt 82 3204 3219 113561011 102 117 GTCTTGATCAATTCTG Deoxy, MOE, and cEt 85 3206 3221 114561012 104 119 TTGTCTTGATCAATTC Deoxy, MOE, and cEt 64 3208 3223 3621561013 106 121 AATTGTCTTGATCAAT Deoxy, MOE, and cEt 21 3210 3225 3622561014 108 123 TGAATTGTCTTGATCA Deoxy, MOE, and cEt 66 3212 3227 3623561015 110 125 GATGAATTGTCTTGAT Deoxy, MOE, and cEt 51 3214 3229 3624561016 112 127 ATGATGAATTGTCTTG Deoxy, MOE, and cEt 71 3216 3231 3625561017 115 130 CAAATGATGAATTGTC Deoxy, MOE, and cEt 36 3219 3234 3626561018 117 132 ATCAAATGATGAATTG Deoxy, MOE, and cEt 27 3221 3236 3627561019 125 140 GATAGAGAATCAAATG Deoxy, MOE, and cEt 11 3229 3244 3628561020 129 144 TGGAGATAGAGAATCA Deoxy, MOE, and cEt 73 3233 3248 3629561021 131 146 TCTGGAGATAGAGAAT Deoxy, MOE, and cEt 51 3235 3250 3630561022 135 150 TGGCTCTGGAGATAGA Deoxy, MOE, and cEt 76 3239 3254 115561023 137 152 TTTGGCTCTGGAGATA Deoxy, MOE, and cEt 73 3241 3256 3631561024 139 154 ATTTTGGCTCTGGAGA Deoxy, MOE, and cEt 61 3243 3258 3632561025 141 156 TGATTTTGGCTCTGGA Deoxy, MOE, and cEt 83 3245 3260 116561026 143 158 CTTGATTTTGGCTCTG Deoxy, MOE, and cEt 83 3247 3262 117561027 145 160 ATCTTGATTTTGGCTC Deoxy, MOE, and cEt 67 3249 3264 3633559277 147 162 AAATCTTGATTTTGGC Deoxy, MOE, and cEt 75 3251 3266 110561028 149 164 GCAAATCTTGATTTTG Deoxy, MOE, and cEt 53 3253 3268 3634561029 151 166 TAGCAAATCTTGATTT Deoxy, MOE, and cEt 27 3255 3270 3635561030 153 168 CATAGCAAATCTTGAT Deoxy, MOE, and cEt 63 3257 3272 3636561031 155 170 AACATAGCAAATCTTG Deoxy, MOE, and cEt 56 3259 3274 3637561032 157 172 CTAACATAGCAAATCT Deoxy, MOE, and cEt 67 3261 3276 3638561033 159 174 GTCTAACATAGCAAAT Deoxy, MOE, and cEt 51 3263 3278 3639561034 174 189 TAAAATTTTTACATCG Deoxy, MOE, and cEt 4 3278 3293 3640561035 177 192 GGCTAAAATTTTTACA Deoxy, MOE, and cEt 0 3281 3296 3641561036 182 197 CCATTGGCTAAAATTT Deoxy, MOE, and cEt 3 3286 3301 3642561037 184 199 GGCCATTGGCTAAAAT Deoxy, MOE, and cEt 16 3288 3303 3643561038 186 201 GAGGCCATTGGCTAAA Deoxy, MOE, and cEt 42 3290 3305 3644561039 188 203 AGGAGGCCATTGGCTA Deoxy, MOE, and cEt 61 3292 3307 3645561040 190 205 GAAGGAGGCCATTGGC Deoxy, MOE, and cEt 35 3294 3309 3646561041 192 207 CTGAAGGAGGCCATTG Deoxy, MOE, and cEt 37 3296 3311 3647561042 194 209 AACTGAAGGAGGCCAT Deoxy, MOE, and cEt 22 3298 3313 3648561043 196 211 CCAACTGAAGGAGGCC Deoxy, MOE, and cEt 33 3300 3315 3649561044 198 213 TCCCAACTGAAGGAGG Deoxy, MOE, and cEt 19 3302 3317 3650561045 200 215 TGTCCCAACTGAAGGA Deoxy, MOE, and cEt 33 3304 3319 3651561046 202 217 CATGTCCCAACTGAAG Deoxy, MOE, and cEt 19 3306 3321 3652561047 204 219 ACCATGTCCCAACTGA Deoxy, MOE, and cEt 19 3308 3323 3653561048 206 221 AGACCATGTCCCAACT Deoxy, MOE, and cEt 19 3310 3325 3654561049 208 223 TAAGACCATGTCCCAA Deoxy, MOE, and cEt 0 3312 3327 3655561050 210 225 TTTAAGACCATGTCCC Deoxy, MOE, and cEt 5 3314 3329 3656561051 212 227 TCTTTAAGACCATGTC Deoxy, MOE, and cEt 10 3316 3331 3657561052 214 229 AGTCTTTAAGACCATG Deoxy, MOE, and cEt 10 3318 3333 3658561053 216 231 AAAGTCTTTAAGACCA Deoxy, MOE, and cEt 29 3320 3335 3659561054 218 233 ACAAAGTCTTTAAGAC Deoxy, MOE, and cEt 19 3322 3337 3660561055 220 235 GGACAAAGTCTTTAAG Deoxy, MOE, and cEt 21 3324 3339 3661561056 222 237 ATGGACAAAGTCTTTA Deoxy, MOE, and cEt 12 3326 3341 3662561057 224 239 TTATGGACAAAGTCTT Deoxy, MOE, and cEt 10 3328 3343 3663561058 226 241 TCTTATGGACAAAGTC Deoxy, MOE, and cEt 9 3330 3345 3664561059 228 243 CGTCTTATGGACAAAG Deoxy, MOE, and cEt 0 3332 3347 3665561060 242 257 TTAATTTGGCCCTTCG Deoxy, MOE, and cEt 28 3346 3361 3666561061 244 259 CATTAATTTGGCCCTT Deoxy, MOE, and cEt 13 3348 3363 3667561062 246 261 GTCATTAATTTGGCCC Deoxy, MOE, and cEt 63 3350 3365 3668561063 248 263 ATGTCATTAATTTGGC Deoxy, MOE, and cEt 37 3352 3367 3669561064 267 282 TATGTTGAGTTTTTGA Deoxy, MOE, and cEt 16 3371 3386 3670561065 272 287 TCAAATATGTTGAGTT Deoxy, MOE, and cEt 21 3376 3391 3671561066 274 289 GATCAAATATGTTGAG Deoxy, MOE, and cEt 36 3378 3393 3672560990 709 724 TTCTTGGTGCTCTTGG Deoxy, MOE, and cEt 73 6722 6737 111337487 804 823 CACTTGTATGTTCACCTCTG 5-10-5 MOE 76 7389 7408 28 5616041850 1865 GTACAATTACCAGTCC Deoxy, MOE, and cEt 59 10822 10837 3673561605 1852 1867 CTGTACAATTACCAGT Deoxy, MOE, and cEt 54 10824 108393674 561606 1854 1869 AACTGTACAATTACCA Deoxy, MOE, and cEt 57 1082610841 3675 561607 1856 1871 AGAACTGTACAATTAC Deoxy, MOE, and cEt 3610828 10843 3676 561608 1858 1873 TAAGAACTGTACAATT Deoxy, MOE, and cEt29 10830 10845 3677 561609 1862 1877 CATTTAAGAACTGTAC Deoxy, MOE, andcEt 24 10834 10849 3678 561610 1870 1885 TACTACAACATTTAAG Deoxy, MOE,and cEt 1 10842 10857 3679 561611 1874 1889 TTAATACTACAACATT Deoxy, MOE,and cEt 0 10846 10861 3680 561612 1880 1895 TTGAAATTAATACTAC Deoxy, MOE,and cEt 6 10852 10867 3681 561613 1883 1898 GTTTTGAAATTAATAC Deoxy, MOE,and cEt 34 10855 10870 3682 561614 1892 1907 CGATTTTTAGTTTTGA Deoxy,MOE, and cEt 22 10864 10879 3683 561615 1894 1909 GACGATTTTTAGTTTTDeoxy, MOE, and cEt 29 10866 10881 3684 561616 1896 1911CTGACGATTTTTAGTT Deoxy, MOE, and cEt 50 10868 10883 3685 561617 18981913 TGCTGACGATTTTTAG Deoxy, MOE, and cEt 54 10870 10885 3686 5616181900 1915 TGTGCTGACGATTTTT Deoxy, MOE, and cEt 70 10872 10887 3687561619 1902 1917 TCTGTGCTGACGATTT Deoxy, MOE, and cEt 69 10874 108893688 561620 1904 1919 ACTCTGTGCTGACGAT Deoxy, MOE, and cEt 78 1087610891 135 561621 1906 1921 ATACTCTGTGCTGACG Deoxy, MOE, and cEt 87 1087810893 134 561622 1908 1923 ACATACTCTGTGCTGA Deoxy, MOE, and cEt 80 1088010895 136 561623 1911 1926 TACACATACTCTGTGC Deoxy, MOE, and cEt 61 1088310898 3689 561624 1913 1928 TTTACACATACTCTGT Deoxy, MOE, and cEt 6810885 10900 3690 561625 1917 1932 GATTTTTACACATACT Deoxy, MOE, and cEt17 10889 10904 3691 561626 1946 1961 GAAGCATCAGTTTAAA Deoxy, MOE, andcEt 27 10918 10933 3692 561627 1948 1963 ATGAAGCATCAGTTTA Deoxy, MOE,and cEt 5 10920 10935 3693 561628 1956 1971 GTAGCAAAATGAAGCA Deoxy, MOE,and cEt 73 10928 10943 137 561629 1958 1973 TTGTAGCAAAATGAAG Deoxy, MOE,and cEt 42 10930 10945 3694 561630 1976 1991 CATTTACTCCAAATTA Deoxy,MOE, and cEt 43 10948 10963 3695 561631 1981 1996 TCAAACATTTACTCCADeoxy, MOE, and cEt 82 10953 10968 138 561632 2006 2021 CATTAGGTTTCATAAADeoxy, MOE, and cEt 19 10978 10993 3696 561633 2008 2023TTCATTAGGTTTCATA Deoxy, MOE, and cEt 15 10980 10995 3697 561634 20102025 GCTTCATTAGGTTTCA Deoxy, MOE, and cEt 57 10982 10997 3698 5616352012 2027 CTGCTTCATTAGGTTT Deoxy, MOE, and cEt 0 10984 10999 3699 5616362014 2029 TTCTGCTTCATTAGGT Deoxy, MOE, and cEt 65 10986 11001 3700561637 2016 2031 AATTCTGCTTCATTAG Deoxy, MOE, and cEt 48 10988 110033701 561638 2024 2039 CAGTATTTAATTCTGC Deoxy, MOE, and cEt 38 1099611011 3702 561639 2039 2054 GAACTTATTTTAATAC Deoxy, MOE, and cEt 2911011 11026 3703 561640 2041 2056 GCGAACTTATTTTAAT Deoxy, MOE, and cEt38 11013 11028 3704 561641 2043 2058 CAGCGAACTTATTTTA Deoxy, MOE, andcEt 46 11015 11030 3705 561642 2045 2060 GACAGCGAACTTATTT Deoxy, MOE,and cEt 64 11017 11032 3706 561643 2047 2062 AAGACAGCGAACTTAT Deoxy,MOE, and cEt 19 11019 11034 3707 561644 2049 2064 TAAAGACAGCGAACTTDeoxy, MOE, and cEt 76 11021 11036 139 561645 2051 2066 TTTAAAGACAGCGAACDeoxy, MOE, and cEt 49 11023 11038 3708 561646 2053 2068TGTTTAAAGACAGCGA Deoxy, MOE, and cEt 81 11025 11040 140 561647 2065 2080GTCATCTCCATTTGTT Deoxy, MOE, and cEt 60 11037 11052 3709 561648 20672082 TAGTCATCTCCATTTG Deoxy, MOE, and cEt 69 11039 11054 3710 5616492069 2084 AGTAGTCATCTCCATT Deoxy, MOE, and cEt 82 11041 11056 141 5616502071 2086 TTAGTAGTCATCTCCA Deoxy, MOE, and cEt 79 11043 11058 142 5616512073 2088 ACTTAGTAGTCATCTC Deoxy, MOE, and cEt 66 11045 11060 3711561652 2075 2090 TGACTTAGTAGTCATC Deoxy, MOE, and cEt 62 11047 110623712 561653 2077 2092 TGTGACTTAGTAGTCA Deoxy, MOE, and cEt 52 1104911064 3713 561654 2079 2094 AATGTGACTTAGTAGT Deoxy, MOE, and cEt 4411051 11066 3714 561655 2081 2096 TCAATGTGACTTAGTA Deoxy, MOE, and cEt65 11053 11068 3715 561656 2083 2098 AGTCAATGTGACTTAG Deoxy, MOE, andcEt 70 11055 11070 3716 561657 2085 2100 AAAGTCAATGTGACTT Deoxy, MOE,and cEt 2 11057 11072 3717 561658 2087 2102 TTAAAGTCAATGTGAC Deoxy, MOE,and cEt 15 11059 11074 3718 561659 2089 2104 TGTTAAAGTCAATGTG Deoxy,MOE, and cEt 27 11061 11076 3719 561660 2091 2106 CATGTTAAAGTCAATGDeoxy, MOE, and cEt 51 11063 11078 3720 561661 2093 2108CTCATGTTAAAGTCAA Deoxy, MOE, and cEt 53 11065 11080 3721 561662 20952110 ACCTCATGTTAAAGTC Deoxy, MOE, and cEt 55 11067 11082 3722 5616632097 2112 ATACCTCATGTTAAAG Deoxy, MOE, and cEt 25 11069 11084 3723561664 2099 2114 TGATACCTCATGTTAA Deoxy, MOE, and cEt 0 11071 11086 3724561665 2101 2116 AGTGATACCTCATGTT Deoxy, MOE, and cEt 38 11073 110883725 561666 2103 2118 ATAGTGATACCTCATG Deoxy, MOE, and cEt 61 1107511090 3726 561667 2105 2120 GTATAGTGATACCTCA Deoxy, MOE, and cEt 6311077 11092 3727 561668 2107 2122 AGGTATAGTGATACCT Deoxy, MOE, and cEt27 11079 11094 3728 561669 2109 2124 TAAGGTATAGTGATAC Deoxy, MOE, andcEt 34 11081 11096 3729 561670 2111 2126 AATAAGGTATAGTGAT Deoxy, MOE,and cEt 22 11083 11098 3730

TABLE 151 Inhibition of ANGPTL3 mRNA by oligonucleotides targeting SEQID NO: 1 and 2 SEQ SEQ SEQ SEQ ID ID ID ID NO: 1 NO: NO: 2 NO: 2 Start 1Stop % Start Stop SEQ ID ISIS NO Site Site Sequence Chemistry inhibitionSite Site NO 562220 N/A N/A GTAAACTTATTGATAA Deoxy, MOE, and cEt 0 76707685 3731 562221 N/A N/A GGCATAGTAAACTTAT Deoxy, MOE, and cEt 22 76767691 3732 562222 N/A N/A AATTTTGGCATAGTAA Deoxy, MOE, and cEt 0 76827697 3733 562223 N/A N/A GGCAATTAATGAATTT Deoxy, MOE, and cEt 15 76937708 3734 562224 N/A N/A GTGAAAGGCAATTAAT Deoxy, MOE, and cEt 7 76997714 3735 562225 N/A N/A AGTTAAGTGAAAGGCA Deoxy, MOE, and cEt 0 77057720 3736 562226 N/A N/A CCCAAAAGTTAAGTGA Deoxy, MOE, and cEt 27 77117726 3737 562227 N/A N/A TATGGTCCCAAAAGTT Deoxy, MOE, and cEt 35 77177732 3738 562228 N/A N/A ATTTATTATGGTCCCA Deoxy, MOE, and cEt 67 77237738 3739 562229 N/A N/A GTTATGGCAATACATT Deoxy, MOE, and cEt 37 77447759 3740 562230 N/A N/A ATTAATGTTATGGCAA Deoxy, MOE, and cEt 33 77507765 3741 562231 N/A N/A GTAGTTTATTAATGTT Deoxy, MOE, and cEt 15 77577772 3742 562232 N/A N/A TGTAAGGTAGTTTATT Deoxy, MOE, and cEt 23 77637778 3743 562233 N/A N/A TGGTTTTGTAAGGTAG Deoxy, MOE, and cEt 43 77697784 3744 562234 N/A N/A AATTGGTGGTTTTGTA Deoxy, MOE, and cEt 18 77757790 3745 562235 N/A N/A GATTTTAATTGGTGGT Deoxy, MOE, and cEt 21 77817796 3746 562236 N/A N/A GATGTAAATAACACTT Deoxy, MOE, and cEt 9 78097824 3747 562237 N/A N/A TTGACAGATGTAAATA Deoxy, MOE, and cEt 11 78157830 3748 562238 N/A N/A TTTATGTTGACAGATG Deoxy, MOE, and cEt 20 78217836 3749 562239 N/A N/A AGTAGATTTATGTTGA Deoxy, MOE, and cEt 9 78277842 3750 562240 N/A N/A CCTGAATATAATGAAT Deoxy, MOE, and cEt 29 78597874 3751 562241 N/A N/A GGACTACCTGAATATA Deoxy, MOE, and cEt 17 78657880 3752 562242 N/A N/A ACCATCAAGCCTCCCA Deoxy, MOE, and cEt 45 79567971 3753 562243 N/A N/A CCCCTTACCATCAAGC Deoxy, MOE, and cEt 31 79627977 3754 562244 N/A N/A TGTAGTCCCCTTACCA Deoxy, MOE, and cEt 16 79687983 3755 562245 N/A N/A ATTGAATGTAGTCCCC Deoxy, MOE, and cEt 19 79747989 3756 562246 N/A N/A GATTAGCAAGTGAATG Deoxy, MOE, and cEt 6 79948009 3757 562247 N/A N/A TTTGTAGATTAGCAAG Deoxy, MOE, and cEt 24 80008015 3758 562248 N/A N/A AAGAGGTTCTCAGTAA Deoxy, MOE, and cEt 28 80198034 3759 562249 N/A N/A GTCCATAAGAGGTTCT Deoxy, MOE, and cEt 34 80258040 3760 562250 N/A N/A TACCTGGTCCATAAGA Deoxy, MOE, and cEt 10 80318046 3761 562251 N/A N/A TCCTAATACCTGGTCC Deoxy, MOE, and cEt 32 80378052 3762 562252 N/A N/A TACTTTTCCTAATACC Deoxy, MOE, and cEt 20 80438058 3763 562253 N/A N/A CGTTACTACTTTTCCT Deoxy, MOE, and cEt 29 80498064 3764 562254 N/A N/A CTGAGACTGCTTCTCG Deoxy, MOE, and cEt 36 80678082 3765 562255 N/A N/A TGAAGGCTGAGACTGC Deoxy, MOE, and cEt 40 80738088 3766 562256 N/A N/A TAAATTATATGAAGGC Deoxy, MOE, and cEt 9 80828097 3767 562257 N/A N/A GTAATTGTTTGATAAT Deoxy, MOE, and cEt 0 80978112 3768 562258 N/A N/A TACTAACAAATGTGTA Deoxy, MOE, and cEt 0 81108125 3769 562259 N/A N/A GTAATTTACTAACAAA Deoxy, MOE, and cEt 0 81168131 3770 562260 N/A N/A ATAAGTGTAATTTACT Deoxy, MOE, and cEt 0 81228137 3771 562261 N/A N/A GTTGTAATAAGTGTAA Deoxy, MOE, and cEt 0 81288143 3772 562262 N/A N/A GTGATAAATATAATTC Deoxy, MOE, and cEt 0 81558170 3773 562263 N/A N/A CATGTAATTGTGATAA Deoxy, MOE, and cEt 20 81648179 3774 562264 N/A N/A GTATATTTAAGAACAG Deoxy, MOE, and cEt 13 81818196 3775 562265 N/A N/A TTGTGATAAGTATATT Deoxy, MOE, and cEt 3 81908205 3776 562266 N/A N/A TGGAATTAAATTGTGA Deoxy, MOE, and cEt 0 82008215 3777 562267 N/A N/A AAGCCGTGGAATTAAA Deoxy, MOE, and cEt 10 82068221 3778 562268 N/A N/A CATTGTAAGCCGTGGA Deoxy, MOE, and cEt 54 82128227 3779 562269 N/A N/A TATGATCATTGTAAGC Deoxy, MOE, and cEt 0 82188233 3780 562270 N/A N/A TATAGTTATGATCATT Deoxy, MOE, and cEt 0 82248239 3781 562271 N/A N/A GACATAACATTTAATC Deoxy, MOE, and cEt 21 82588273 3782 562272 N/A N/A ACTTATGACATAACAT Deoxy, MOE, and cEt 14 82648279 3783 562273 N/A N/A GTTACTACTTATGACA Deoxy, MOE, and cEt 30 82708285 3784 562274 N/A N/A GTAACAGTTACTACTT Deoxy, MOE, and cEt 24 82768291 3785 562275 N/A N/A GCTTATTTGTAACAGT Deoxy, MOE, and cEt 17 82848299 3786 562276 N/A N/A TTCACAGCTTATTTGT Deoxy, MOE, and cEt 20 82908305 3787 562277 N/A N/A GTTCTTTTCACAGCTT Deoxy, MOE, and cEt 46 82968311 3788 562278 N/A N/A GGAGTGGTTCTTTTCA Deoxy, MOE, and cEt 35 83028317 3789 562279 N/A N/A ATGCTAGGAGTGGTTC Deoxy, MOE, and cEt 29 83088323 3790 562280 N/A N/A TGACTAATGCTAGGAG Deoxy, MOE, and cEt 4 83148329 3791 562281 N/A N/A ATAGAGTGACTAATGC Deoxy, MOE, and cEt 23 83208335 3792 562282 N/A N/A GAGAGAATAGAGTGAC Deoxy, MOE, and cEt 15 83268341 3793 562284 N/A N/A ATTGATATGTAAAACG Deoxy, MOE, and cEt 7 83478362 3794 562285 N/A N/A CAATTAATTGATATGT Deoxy, MOE, and cEt 14 83538368 3795 562286 N/A N/A CCTTTTAACTTCCAAT Deoxy, MOE, and cEt 40 83658380 3796 562287 N/A N/A CCTGGTCCTTTTAACT Deoxy, MOE, and cEt 29 83718386 3797 562288 N/A N/A GAGTTTCCTGGTCCTT Deoxy, MOE, and cEt 49 83778392 3798 562289 N/A N/A ATGTCTGAGTTTCCTG Deoxy, MOE, and cEt 16 83838398 3799 562290 N/A N/A TACTGTATGTCTGAGT Deoxy, MOE, and cEt 33 83898404 3800 562291 N/A N/A CCATACATTCTATATA Deoxy, MOE, and cEt 10 84378452 3801 562292 N/A N/A TATAAGCCATACATTC Deoxy, MOE, and cEt 24 84438458 3802 562293 N/A N/A ATTCATTATAAGCCAT Deoxy, MOE, and cEt 38 84498464 3803 562295 N/A N/A CATTGAGTTAACTAAT Deoxy, MOE, and cEt 7 84638478 3804 562296 N/A N/A AATTTGCATTGAGTTA Deoxy, MOE, and cEt 18 84698484 3805 561144 525 540 TGAAGTTACTTCTGGG Deoxy, MOE, and cEt 39 36293644 3806 561145 527 542 AGTGAAGTTACTTCTG Deoxy, MOE, and cEt 51 36313646 3807 561146 529 544 TAAGTGAAGTTACTTC Deoxy, MOE, and cEt 40 36333648 3808 561147 533 548 GTTTTAAGTGAAGTTA Deoxy, MOE, and cEt 29 N/A N/A3809 561148 535 550 AAGTTTTAAGTGAAGT Deoxy, MOE, and cEt 19 N/A N/A 3810561149 547 562 GTTTTTCTACAAAAGT Deoxy, MOE, and cEt 38 4285 4300 3811561150 560 575 ATGCTATTATCTTGTT Deoxy, MOE, and cEt 30 4298 4313 3812561151 562 577 TGATGCTATTATCTTG Deoxy, MOE, and cEt 36 4300 4315 3813561152 564 579 TTTGATGCTATTATCT Deoxy, MOE, and cEt 23 4302 4317 3814561153 567 582 GTCTTTGATGCTATTA Deoxy, MOE, and cEt 51 4305 4320 3815561154 569 584 AGGTCTTTGATGCTAT Deoxy, MOE, and cEt 60 4307 4322 3816561155 571 586 GAAGGTCTTTGATGCT Deoxy, MOE, and cEt 61 4309 4324 3817561156 573 588 GAGAAGGTCTTTGATG Deoxy, MOE, and cEt 30 4311 4326 3818561157 575 590 TGGAGAAGGTCTTTGA Deoxy, MOE, and cEt 40 4313 4328 3819561158 577 592 TCTGGAGAAGGTCTTT Deoxy, MOE, and cEt 46 4315 4330 3820561159 579 594 GGTCTGGAGAAGGTCT Deoxy, MOE, and cEt 57 4317 4332 3821561160 581 596 ACGGTCTGGAGAAGGT Deoxy, MOE, and cEt 57 4319 4334 3822561161 583 598 CCACGGTCTGGAGAAG Deoxy, MOE, and cEt 56 4321 4336 3823561162 585 600 TTCCACGGTCTGGAGA Deoxy, MOE, and cEt 50 4323 4338 3824561163 587 602 TCTTCCACGGTCTGGA Deoxy, MOE, and cEt 77 4325 4340 3825561164 589 604 GGTCTTCCACGGTCTG Deoxy, MOE, and cEt 89 4327 4342 3826561165 591 606 TTGGTCTTCCACGGTC Deoxy, MOE, and cEt 79 4329 4344 3827561166 593 608 TATTGGTCTTCCACGG Deoxy, MOE, and cEt 39 4331 4346 3828561167 595 610 TATATTGGTCTTCCAC Deoxy, MOE, and cEt 22 4333 4348 3829561168 597 612 TTTATATTGGTCTTCC Deoxy, MOE, and cEt 43 4335 4350 3830561169 599 614 TGTTTATATTGGTCTT Deoxy, MOE, and cEt 50 4337 4352 3831561170 601 616 ATTGTTTATATTGGTC Deoxy, MOE, and cEt 27 4339 4354 3832561171 603 618 TAATTGTTTATATTGG Deoxy, MOE, and cEt 21 4341 4356 3833561172 607 622 GGTTTAATTGTTTATA Deoxy, MOE, and cEt 22 4345 4360 3834561173 610 625 GTTGGTTTAATTGTTT Deoxy, MOE, and cEt 33 4348 4363 3835561174 612 627 CTGTTGGTTTAATTGT Deoxy, MOE, and cEt 13 4350 4365 3836561175 614 629 TGCTGTTGGTTTAATT Deoxy, MOE, and cEt 26 4352 4367 3837561176 616 631 TATGCTGTTGGTTTAA Deoxy, MOE, and cEt 40 4354 4369 3838561177 618 633 ACTATGCTGTTGGTTT Deoxy, MOE, and cEt 68 4356 4371 3839561178 620 635 TGACTATGCTGTTGGT Deoxy, MOE, and cEt 64 4358 4373 3840561179 622 637 TTTGACTATGCTGTTG Deoxy, MOE, and cEt 42 4360 4375 3841561180 624 639 TATTTGACTATGCTGT Deoxy, MOE, and cEt 16 4362 4377 3842561181 626 641 TTTATTTGACTATGCT Deoxy, MOE, and cEt 17 4364 4379 3843561182 628 643 CTTTTATTTGACTATG Deoxy, MOE, and cEt 7 4366 4381 3844561183 645 660 GAGCTGATTTTCTATT Deoxy, MOE, and cEt 18 N/A N/A 3845561184 647 662 CTGAGCTGATTTTCTA Deoxy, MOE, and cEt 42 N/A N/A 3846561185 649 664 TTCTGAGCTGATTTTC Deoxy, MOE, and cEt 32 N/A N/A 3847561186 651 666 CCTTCTGAGCTGATTT Deoxy, MOE, and cEt 14 N/A N/A 3848561187 653 668 GTCCTTCTGAGCTGAT Deoxy, MOE, and cEt 39 6666 6681 3849561188 655 670 TAGTCCTTCTGAGCTG Deoxy, MOE, and cEt 7 6668 6683 3850561189 657 672 ACTAGTCCTTCTGAGC Deoxy, MOE, and cEt 32 6670 6685 3851561190 659 674 ATACTAGTCCTTCTGA Deoxy, MOE, and cEt 19 6672 6687 3852561191 661 676 GAATACTAGTCCTTCT Deoxy, MOE, and cEt 37 6674 6689 3853561192 663 678 TTGAATACTAGTCCTT Deoxy, MOE, and cEt 50 6676 6691 3854561193 665 680 TCTTGAATACTAGTCC Deoxy, MOE, and cEt 28 6678 6693 3855561194 667 682 GTTCTTGAATACTAGT Deoxy, MOE, and cEt 34 6680 6695 3856561195 669 684 GGGTTCTTGAATACTA Deoxy, MOE, and cEt 61 6682 6697 3857561196 671 686 GTGGGTTCTTGAATAC Deoxy, MOE, and cEt 21 6684 6699 3858561197 673 688 CTGTGGGTTCTTGAAT Deoxy, MOE, and cEt 45 6686 6701 3859561198 675 690 TTCTGTGGGTTCTTGA Deoxy, MOE, and cEt 0 6688 6703 3860561199 679 694 AAATTTCTGTGGGTTC Deoxy, MOE, and cEt 31 6692 6707 3861561200 681 696 AGAAATTTCTGTGGGT Deoxy, MOE, and cEt 60 6694 6709 3862561201 684 699 TAGAGAAATTTCTGTG Deoxy, MOE, and cEt 35 6697 6712 3863561202 686 701 GATAGAGAAATTTCTG Deoxy, MOE, and cEt 36 6699 6714 3864561203 694 709 GCTTGGAAGATAGAGA Deoxy, MOE, and cEt 39 6707 6722 3865561204 696 711 TGGCTTGGAAGATAGA Deoxy, MOE, and cEt 32 6709 6724 3866561205 698 713 CTTGGCTTGGAAGATA Deoxy, MOE, and cEt 23 6711 6726 3867561206 700 715 CTCTTGGCTTGGAAGA Deoxy, MOE, and cEt 21 6713 6728 3868561207 702 717 TGCTCTTGGCTTGGAA Deoxy, MOE, and cEt 34 6715 6730 3869561208 704 719 GGTGCTCTTGGCTTGG Deoxy, MOE, and cEt 71 6717 6732 118561209 706 721 TTGGTGCTCTTGGCTT Deoxy, MOE, and cEt 59 6719 6734 3870561210 708 723 TCTTGGTGCTCTTGGC Deoxy, MOE, and cEt 65 6721 6736 3871560990 709 724 TTCTTGGTGCTCTTGG Deoxy, MOE, and cEt 54 6722 6737 111561211 710 725 GTTCTTGGTGCTCTTG Deoxy, MOE, and cEt 60 6723 6738 3872561212 712 727 TAGTTCTTGGTGCTCT Deoxy, MOE, and cEt 53 6725 6740 3873561213 714 729 AGTAGTTCTTGGTGCT Deoxy, MOE, and cEt 50 6727 6742 3874561214 716 731 GGAGTAGTTCTTGGTG Deoxy, MOE, and cEt 31 6729 6744 3875561215 718 733 AGGGAGTAGTTCTTGG Deoxy, MOE, and cEt 0 6731 6746 3876561216 720 735 AAAGGGAGTAGTTCTT Deoxy, MOE, and cEt 25 6733 6748 3877561217 722 737 AGAAAGGGAGTAGTTC Deoxy, MOE, and cEt 28 6735 6750 3878561218 724 739 GAAGAAAGGGAGTAGT Deoxy, MOE, and cEt 10 6737 6752 3879561219 726 741 CTGAAGAAAGGGAGTA Deoxy, MOE, and cEt 47 6739 6754 3880561220 730 745 TCAACTGAAGAAAGGG Deoxy, MOE, and cEt 50 6743 6758 3881337487 804 823 CACTTGTATGTTCACCTCTG 5-10-5 MOE 52 7389 7408 28 561297926 941 TCATTGAAGTTTTGTG Deoxy, MOE, and cEt 28 7913 7928 3882 561298930 945 CGTTTCATTGAAGTTT Deoxy, MOE, and cEt 35 7917 7932 3883 561299944 959 TTGTAGTTCTCCCACG Deoxy, MOE, and cEt 30 7931 7946 3884 561300946 961 ATTTGTAGTTCTCCCA Deoxy, MOE, and cEt 32 7933 7948 3885 561301948 963 ATATTTGTAGTTCTCC Deoxy, MOE, and cEt 24 7935 7950 3886 561302950 965 CCATATTTGTAGTTCT Deoxy, MOE, and cEt 5 7937 7952 3887 561303 952967 AACCATATTTGTAGTT Deoxy, MOE, and cEt 3 7939 7954 3888 561304 956 971CCAAAACCATATTTGT Deoxy, MOE, and cEt 19 7943 7958 3889 561305 959 974CTCCCAAAACCATATT Deoxy, MOE, and cEt 23 7946 7961 3890 561306 961 976GCCTCCCAAAACCATA Deoxy, MOE, and cEt 25 7948 7963 3891 561307 963 978AAGCCTCCCAAAACCA Deoxy, MOE, and cEt 30 7950 7965 3892 561308 965 980TCAAGCCTCCCAAAAC Deoxy, MOE, and cEt 16 7952 7967 3893 561309 969 984TCCATCAAGCCTCCCA Deoxy, MOE, and cEt 46 N/A N/A 3894 561310 971 986TCTCCATCAAGCCTCC Deoxy, MOE, and cEt 13 N/A N/A 3895 561311 973 988ATTCTCCATCAAGCCT Deoxy, MOE, and cEt 16 N/A N/A 3896 561312 975 990AAATTCTCCATCAAGC Deoxy, MOE, and cEt 20 N/A N/A 3897 561313 979 994ACCAAAATTCTCCATC Deoxy, MOE, and cEt 18 N/A N/A 3898 561314 981 996CAACCAAAATTCTCCA Deoxy, MOE, and cEt 26 N/A N/A 3899 561315 983 998CCCAACCAAAATTCTC Deoxy, MOE, and cEt 38 9558 9573 3900 559316 985 1000GGCCCAACCAAAATTC Deoxy, MOE, and cEt 14 9560 9575 3901 561316 987 1002TAGGCCCAACCAAAAT Deoxy, MOE, and cEt 38 9562 9577 3902 561317 989 1004TCTAGGCCCAACCAAA Deoxy, MOE, and cEt 51 9564 9579 3903 561318 991 1006TCTCTAGGCCCAACCA Deoxy, MOE, and cEt 35 9566 9581 3904 561319 993 1008CTTCTCTAGGCCCAAC Deoxy, MOE, and cEt 31 9568 9583 3905 561320 995 1010ATCTTCTCTAGGCCCA Deoxy, MOE, and cEt 68 9570 9585 119 561321 997 1012ATATCTTCTCTAGGCC Deoxy, MOE, and cEt 30 9572 9587 3906 561322 999 1014GTATATCTTCTCTAGG Deoxy, MOE, and cEt 25 9574 9589 3907 561323 1001 1016GAGTATATCTTCTCTA Deoxy, MOE, and cEt 26 9576 9591 3908 561324 1003 1018TGGAGTATATCTTCTC Deoxy, MOE, and cEt 46 9578 9593 3909 561325 1005 1020TATGGAGTATATCTTC Deoxy, MOE, and cEt 20 9580 9595 3910 561326 1007 1022ACTATGGAGTATATCT Deoxy, MOE, and cEt 20 9582 9597 3911 561327 1009 1024TCACTATGGAGTATAT Deoxy, MOE, and cEt 22 9584 9599 3912 561328 1011 1026CTTCACTATGGAGTAT Deoxy, MOE, and cEt 33 9586 9601 3913 561329 1013 1028TGCTTCACTATGGAGT Deoxy, MOE, and cEt 50 9588 9603 3914 561330 1015 1030ATTGCTTCACTATGGA Deoxy, MOE, and cEt 43 9590 9605 3915 561331 1017 1032AGATTGCTTCACTATG Deoxy, MOE, and cEt 31 9592 9607 3916 561332 1019 1034TTAGATTGCTTCACTA Deoxy, MOE, and cEt 36 9594 9609 3917 561333 1021 1036AATTAGATTGCTTCAC Deoxy, MOE, and cEt 17 9596 9611 3918 561334 1023 1038ATAATTAGATTGCTTC Deoxy, MOE, and cEt 23 9598 9613 3919 561335 1025 1040ACATAATTAGATTGCT Deoxy, MOE, and cEt 13 9600 9615 3920 561336 1031 1046CGTAAAACATAATTAG Deoxy, MOE, and cEt 25 9606 9621 3921 561337 1045 1060CTTCCAACTCAATTCG Deoxy, MOE, and cEt 0 9620 9635 3922 561338 1047 1062GTCTTCCAACTCAATT Deoxy, MOE, and cEt 0 9622 9637 3923 561339 1049 1064CAGTCTTCCAACTCAA Deoxy, MOE, and cEt 15 9624 9639 3924 561340 1051 1066TCCAGTCTTCCAACTC Deoxy, MOE, and cEt 22 9626 9641 3925 561341 1053 1068TTTCCAGTCTTCCAAC Deoxy, MOE, and cEt 2 9628 9643 3926 561342 1056 1071GTCTTTCCAGTCTTCC Deoxy, MOE, and cEt 45 9631 9646 3927 561343 1059 1074GTTGTCTTTCCAGTCT Deoxy, MOE, and cEt 67 9634 9649 120 561344 1061 1076TTGTTGTCTTTCCAGT Deoxy, MOE, and cEt 43 9636 9651 3928 561345 1063 1078GTTTGTTGTCTTTCCA Deoxy, MOE, and cEt 57 9638 9653 121 561346 1068 1083ATAATGTTTGTTGTCT Deoxy, MOE, and cEt 6 9643 9658 3929 561347 1098 1113GTGATTTCCCAAGTAA Deoxy, MOE, and cEt 66 9673 9688 122 561348 1113 1128CGTATAGTTGGTTTCG Deoxy, MOE, and cEt 54 9688 9703 3930 561349 1127 1142GCAACTAGATGTAGCG Deoxy, MOE, and cEt 50 9702 9717 3931 561350 1129 1144TCGCAACTAGATGTAG Deoxy, MOE, and cEt 9 9704 9719 3932 561351 1131 1146AATCGCAACTAGATGT Deoxy, MOE, and cEt 9 9706 9721 3933 561352 1133 1148GTAATCGCAACTAGAT Deoxy, MOE, and cEt 15 9708 9723 3934 561353 1135 1150CAGTAATCGCAACTAG Deoxy, MOE, and cEt 41 9710 9725 3935 561354 1137 1152GCCAGTAATCGCAACT Deoxy, MOE, and cEt 38 9712 9727 3936 561355 1139 1154TTGCCAGTAATCGCAA Deoxy, MOE, and cEt 32 9714 9729 3937 561356 1141 1156CATTGCCAGTAATCGC Deoxy, MOE, and cEt 54 9716 9731 3938 561357 1143 1158GACATTGCCAGTAATC Deoxy, MOE, and cEt 20 9718 9733 3939 561358 1145 1160GGGACATTGCCAGTAA Deoxy, MOE, and cEt 0 9720 9735 3940 561359 1160 1175TCCGGGATTGCATTGG Deoxy, MOE, and cEt 43 9735 9750 3941 561360 1162 1177TTTCCGGGATTGCATT Deoxy, MOE, and cEt 31 9737 9752 3942 561361 1164 1179GTTTTCCGGGATTGCA Deoxy, MOE, and cEt 31 9739 9754 3943 561362 1166 1181TTGTTTTCCGGGATTG Deoxy, MOE, and cEt 36 9741 9756 3944 561363 1168 1183CTTTGTTTTCCGGGAT Deoxy, MOE, and cEt 22 9743 9758 3945 561364 1170 1185ATCTTTGTTTTCCGGG Deoxy, MOE, and cEt 13 9745 9760 3946 561365 1172 1187AAATCTTTGTTTTCCG Deoxy, MOE, and cEt 7 9747 9762 3947 561366 1177 1192ACACCAAATCTTTGTT Deoxy, MOE, and cEt 8 9752 9767 3948 561367 1179 1194AAACACCAAATCTTTG Deoxy, MOE, and cEt 11 9754 9769 3949 561368 1187 1202CAAGTAGAAAACACCA Deoxy, MOE, and cEt 16 9762 9777 3950 561369 1189 1204CCCAAGTAGAAAACAC Deoxy, MOE, and cEt 23 9764 9779 3951 561370 1191 1206ATCCCAAGTAGAAAAC Deoxy, MOE, and cEt 27 9766 9781 3952 561371 1193 1208TGATCCCAAGTAGAAA Deoxy, MOE, and cEt 25 9768 9783 3953 561372 1195 1210TGTGATCCCAAGTAGA Deoxy, MOE, and cEt 45 9770 9785 3954

TABLE 152 Inhibition of ANGPTL3 mRNA by oligonucleotides targeting SEQID NO: 1 and 2 SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: NO: 2 NO: 2 Start 1Stop % Start Stop SEQ ID ISIS NO Site Site Sequence Chemistry inhibitionSite Site NO 561067 276 291 CTGATCAAATATGTTG Deoxy, MOE, and cEt 54 33803395 3955 561068 278 293 GACTGATCAAATATGT Deoxy, MOE, and cEt 19 33823397 3956 561069 280 295 AAGACTGATCAAATAT Deoxy, MOE, and cEt 17 33843399 3957 561070 286 301 CATAAAAAGACTGATC Deoxy, MOE, and cEt 18 33903405 3958 561071 289 304 GATCATAAAAAGACTG Deoxy, MOE, and cEt 11 33933408 3959 561072 291 306 TAGATCATAAAAAGAC Deoxy, MOE, and cEt 0 33953410 3960 561073 293 308 GATAGATCATAAAAAG Deoxy, MOE, and cEt 15 33973412 3961 561074 295 310 GCGATAGATCATAAAA Deoxy, MOE, and cEt 39 33993414 3962 561075 297 312 CAGCGATAGATCATAA Deoxy, MOE, and cEt 53 34013416 3963 561076 299 314 TGCAGCGATAGATCAT Deoxy, MOE, and cEt 70 34033418 159 561077 301 316 TTTGCAGCGATAGATC Deoxy, MOE, and cEt 60 34053420 3964 561078 303 318 GGTTTGCAGCGATAGA Deoxy, MOE, and cEt 63 34073422 3965 561079 305 320 CTGGTTTGCAGCGATA Deoxy, MOE, and cEt 76 34093424 160 561080 307 322 CACTGGTTTGCAGCGA Deoxy, MOE, and cEt 65 34113426 3966 561081 309 324 TTCACTGGTTTGCAGC Deoxy, MOE, and cEt 45 34133428 3967 561082 311 326 ATTTCACTGGTTTGCA Deoxy, MOE, and cEt 56 34153430 3968 561083 313 328 TGATTTCACTGGTTTG Deoxy, MOE, and cEt 65 34173432 3969 561084 316 331 CTTTGATTTCACTGGT Deoxy, MOE, and cEt 73 34203435 161 561085 341 356 GTTCTTCTCAGTTCCT Deoxy, MOE, and cEt 79 34453460 162 561086 343 358 TAGTTCTTCTCAGTTC Deoxy, MOE, and cEt 50 34473462 3970 561087 345 360 TGTAGTTCTTCTCAGT Deoxy, MOE, and cEt 42 34493464 3971 561088 347 362 TATGTAGTTCTTCTCA Deoxy, MOE, and cEt 27 34513466 3972 561089 349 364 TATATGTAGTTCTTCT Deoxy, MOE, and cEt 37 34533468 3973 561090 352 367 GTTTATATGTAGTTCT Deoxy, MOE, and cEt 39 34563471 3974 561091 355 370 GTAGTTTATATGTAGT Deoxy, MOE, and cEt 55 34593474 3975 561092 358 373 CTTGTAGTTTATATGT Deoxy, MOE, and cEt 48 34623477 3976 561093 360 375 GACTTGTAGTTTATAT Deoxy, MOE, and cEt 43 34643479 3977 561094 362 377 TTGACTTGTAGTTTAT Deoxy, MOE, and cEt 35 34663481 3978 561095 365 380 TTTTTGACTTGTAGTT Deoxy, MOE, and cEt 37 34693484 3979 561096 367 382 CATTTTTGACTTGTAG Deoxy, MOE, and cEt 34 34713486 3980 561097 373 388 CCTCTTCATTTTTGAC Deoxy, MOE, and cEt 48 34773492 3981 561098 386 401 GACATATTCTTTACCT Deoxy, MOE, and cEt 40 34903505 3982 561099 388 403 GTGACATATTCTTTAC Deoxy, MOE, and cEt 43 34923507 3983 561100 393 408 TTCAAGTGACATATTC Deoxy, MOE, and cEt 51 34973512 3984 561101 395 410 AGTTCAAGTGACATAT Deoxy, MOE, and cEt 27 34993514 3985 561102 397 412 TGAGTTCAAGTGACAT Deoxy, MOE, and cEt 63 35013516 3986 561103 399 414 GTTGAGTTCAAGTGAC Deoxy, MOE, and cEt 48 35033518 3987 561104 401 416 GAGTTGAGTTCAAGTG Deoxy, MOE, and cEt 57 35053520 3988 561105 403 418 TTGAGTTGAGTTCAAG Deoxy, MOE, and cEt 32 35073522 3989 561106 405 420 TTTTGAGTTGAGTTCA Deoxy, MOE, and cEt 47 35093524 3990 561107 407 422 AGTTTTGAGTTGAGTT Deoxy, MOE, and cEt 46 35113526 3991 561108 409 424 CAAGTTTTGAGTTGAG Deoxy, MOE, and cEt 48 35133528 3992 561109 411 426 TTCAAGTTTTGAGTTG Deoxy, MOE, and cEt 17 35153530 3993 561110 413 428 CTTTCAAGTTTTGAGT Deoxy, MOE, and cEt 48 35173532 3994 561111 415 430 GGCTTTCAAGTTTTGA Deoxy, MOE, and cEt 56 35193534 3995 561112 417 432 GAGGCTTTCAAGTTTT Deoxy, MOE, and cEt 39 35213536 3996 561113 419 434 AGGAGGCTTTCAAGTT Deoxy, MOE, and cEt 49 35233538 3997 561114 421 436 CTAGGAGGCTTTCAAG Deoxy, MOE, and cEt 49 35253540 3998 561115 423 438 TTCTAGGAGGCTTTCA Deoxy, MOE, and cEt 40 35273542 3999 561116 425 440 TCTTCTAGGAGGCTTT Deoxy, MOE, and cEt 66 35293544 4000 561117 427 442 TTTCTTCTAGGAGGCT Deoxy, MOE, and cEt 74 35313546 4001 561118 442 457 GTTGAAGTAGAATTTT Deoxy, MOE, and cEt 40 35463561 4002 561119 469 484 GTTGCTCTTCTAAATA Deoxy, MOE, and cEt 44 35733588 4003 561120 471 486 TAGTTGCTCTTCTAAA Deoxy, MOE, and cEt 19 35753590 4004 561121 473 488 GTTAGTTGCTCTTCTA Deoxy, MOE, and cEt 67 35773592 4005 561122 475 490 TAGTTAGTTGCTCTTC Deoxy, MOE, and cEt 51 35793594 4006 561123 477 492 GTTAGTTAGTTGCTCT Deoxy, MOE, and cEt 73 35813596 163 561124 479 494 AAGTTAGTTAGTTGCT Deoxy, MOE, and cEt 51 35833598 4007 561125 481 496 TTAAGTTAGTTAGTTG Deoxy, MOE, and cEt 33 35853600 4008 561126 483 498 AATTAAGTTAGTTAGT Deoxy, MOE, and cEt 0 35873602 4009 561127 485 500 TGAATTAAGTTAGTTA Deoxy, MOE, and cEt 5 35893604 4010 561128 487 502 TTTGAATTAAGTTAGT Deoxy, MOE, and cEt 18 35913606 4011 561129 494 509 GGTTGATTTTGAATTA Deoxy, MOE, and cEt 20 35983613 4012 561130 496 511 CAGGTTGATTTTGAAT Deoxy, MOE, and cEt 27 36003615 4013 561131 498 513 TTCAGGTTGATTTTGA Deoxy, MOE, and cEt 33 36023617 4014 561132 500 515 GTTTCAGGTTGATTTT Deoxy, MOE, and cEt 38 36043619 4015 561133 502 517 GAGTTTCAGGTTGATT Deoxy, MOE, and cEt 33 36063621 4016 561134 504 519 TGGAGTTTCAGGTTGA Deoxy, MOE, and cEt 67 36083623 4017 561135 507 522 TTCTGGAGTTTCAGGT Deoxy, MOE, and cEt 32 36113626 4018 561136 509 524 TGTTCTGGAGTTTCAG Deoxy, MOE, and cEt 14 36133628 4019 561137 511 526 GGTGTTCTGGAGTTTC Deoxy, MOE, and cEt 23 36153630 4020 561138 513 528 TGGGTGTTCTGGAGTT Deoxy, MOE, and cEt 30 36173632 4021 561139 515 530 TCTGGGTGTTCTGGAG Deoxy, MOE, and cEt 24 36193634 4022 561140 517 532 CTTCTGGGTGTTCTGG Deoxy, MOE, and cEt 17 36213636 4023 561141 519 534 TACTTCTGGGTGTTCT Deoxy, MOE, and cEt 10 36233638 4024 561142 521 536 GTTACTTCTGGGTGTT Deoxy, MOE, and cEt 11 36253640 4025 561143 523 538 AAGTTACTTCTGGGTG Deoxy, MOE, and cEt 15 36273642 4026 560990 709 724 TTCTTGGTGCTCTTGG Deoxy, MOE, and cEt 79 67226737 111 561221 758 773 CCATCATGTTTTACAT Deoxy, MOE, and cEt 17 67716786 4027 561222 760 775 TGCCATCATGTTTTAC Deoxy, MOE, and cEt 22 N/A N/A4028 561223 763 778 GAATGCCATCATGTTT Deoxy, MOE, and cEt 12 N/A N/A 4029561224 765 780 AGGAATGCCATCATGT Deoxy, MOE, and cEt 26 N/A N/A 4030561225 767 782 GCAGGAATGCCATCAT Deoxy, MOE, and cEt 32 N/A N/A 4031561226 769 784 CAGCAGGAATGCCATC Deoxy, MOE, and cEt 29 N/A N/A 4032561227 771 786 TTCAGCAGGAATGCCA Deoxy, MOE, and cEt 22 N/A N/A 4033561228 773 788 CATTCAGCAGGAATGC Deoxy, MOE, and cEt 23 7358 7373 4034561229 775 790 TACATTCAGCAGGAAT Deoxy, MOE, and cEt 28 7360 7375 4035561230 777 792 GGTACATTCAGCAGGA Deoxy, MOE, and cEt 61 7362 7377 4036561231 779 794 GTGGTACATTCAGCAG Deoxy, MOE, and cEt 57 7364 7379 4037561232 781 796 TGGTGGTACATTCAGC Deoxy, MOE, and cEt 59 7366 7381 4038561233 787 802 TATAAATGGTGGTACA Deoxy, MOE, and cEt 51 7372 7387 4039561234 789 804 GTTATAAATGGTGGTA Deoxy, MOE, and cEt 50 7374 7389 4040561235 791 806 CTGTTATAAATGGTGG Deoxy, MOE, and cEt 49 7376 7391 4041561236 793 808 CTCTGTTATAAATGGT Deoxy, MOE, and cEt 39 7378 7393 4042561237 795 810 ACCTCTGTTATAAATG Deoxy, MOE, and cEt 47 7380 7395 4043561238 797 812 TCACCTCTGTTATAAA Deoxy, MOE, and cEt 44 7382 7397 4044561239 799 814 GTTCACCTCTGTTATA Deoxy, MOE, and cEt 43 7384 7399 4045561240 801 816 ATGTTCACCTCTGTTA Deoxy, MOE, and cEt 59 7386 7401 4046561241 803 818 GTATGTTCACCTCTGT Deoxy, MOE, and cEt 69 7388 7403 164337487 804 823 CACTTGTATGTTCACCTCTG 5-10-5 MOE 74 7389 7408 28 561242805 820 TTGTATGTTCACCTCT Deoxy, MOE, and cEt 63 7390 7405 4047 561243807 822 ACTTGTATGTTCACCT Deoxy, MOE, and cEt 63 7392 7407 4048 561244809 824 CCACTTGTATGTTCAC Deoxy, MOE, and cEt 57 7394 7409 4049 561245811 826 TGCCACTTGTATGTTC Deoxy, MOE, and cEt 36 7396 7411 4050 561246813 828 CATGCCACTTGTATGT Deoxy, MOE, and cEt 33 7398 7413 4051 561247815 830 TACATGCCACTTGTAT Deoxy, MOE, and cEt 37 7400 7415 4052 561248817 832 CATACATGCCACTTGT Deoxy, MOE, and cEt 36 7402 7417 4053 561249819 834 GGCATACATGCCACTT Deoxy, MOE, and cEt 20 7404 7419 4054 561250821 836 ATGGCATACATGCCAC Deoxy, MOE, and cEt 0 7406 7421 4055 561251 823838 TGATGGCATACATGCC Deoxy, MOE, and cEt 22 7408 7423 4056 561252 825840 TCTGATGGCATACATG Deoxy, MOE, and cEt 34 7410 7425 4057 561253 827842 GGTCTGATGGCATACA Deoxy, MOE, and cEt 46 7412 7427 4058 561254 829844 TGGGTCTGATGGCATA Deoxy, MOE, and cEt 51 7414 7429 4059 561255 834849 GTTGCTGGGTCTGATG Deoxy, MOE, and cEt 45 7419 7434 4060 561256 836851 GAGTTGCTGGGTCTGA Deoxy, MOE, and cEt 70 7421 7436 165 561257 838 853GAGAGTTGCTGGGTCT Deoxy, MOE, and cEt 57 7423 7438 4061 561258 840 855TTGAGAGTTGCTGGGT Deoxy, MOE, and cEt 47 7425 7440 4062 561259 842 857ACTTGAGAGTTGCTGG Deoxy, MOE, and cEt 53 7427 7442 4063 561260 844 859AAACTTGAGAGTTGCT Deoxy, MOE, and cEt 71 7429 7444 166 561261 846 861AAAAACTTGAGAGTTG Deoxy, MOE, and cEt 23 7431 7446 4064 561262 848 863TGAAAAACTTGAGAGT Deoxy, MOE, and cEt 11 7433 7448 4065 561263 850 865CATGAAAAACTTGAGA Deoxy, MOE, and cEt 34 7435 7450 4066 561264 852 867GACATGAAAAACTTGA Deoxy, MOE, and cEt 25 7437 7452 4067 561265 860 875TCACAGTAGACATGAA Deoxy, MOE, and cEt 16 7445 7460 4068 561266 862 877CATCACAGTAGACATG Deoxy, MOE, and cEt 37 7447 7462 4069 561267 864 879AACATCACAGTAGACA Deoxy, MOE, and cEt 57 7449 7464 4070 561268 866 881ATAACATCACAGTAGA Deoxy, MOE, and cEt 40 7451 7466 4071 561269 868 883ATATAACATCACAGTA Deoxy, MOE, and cEt 26 7453 7468 4072 561270 870 885TGATATAACATCACAG Deoxy, MOE, and cEt 35 7455 7470 4073 561271 872 887CCTGATATAACATCAC Deoxy, MOE, and cEt 60 7457 7472 4074 561272 874 889TACCTGATATAACATC Deoxy, MOE, and cEt 37 7459 7474 4075 561273 876 891ACTACCTGATATAACA Deoxy, MOE, and cEt 24 N/A N/A 4076 561274 878 893GGACTACCTGATATAA Deoxy, MOE, and cEt 7 N/A N/A 4077 561275 880 895ATGGACTACCTGATAT Deoxy, MOE, and cEt 33 N/A N/A 4078 561276 882 897CCATGGACTACCTGAT Deoxy, MOE, and cEt 52 N/A N/A 4079 561277 884 899GTCCATGGACTACCTG Deoxy, MOE, and cEt 71 7871 7886 167 561278 886 901ATGTCCATGGACTACC Deoxy, MOE, and cEt 67 7873 7888 4080 561279 888 903TAATGTCCATGGACTA Deoxy, MOE, and cEt 44 7875 7890 4081 559390 890 905ATTAATGTCCATGGAC Deoxy, MOE, and cEt 28 7877 7892 4082 561280 892 907GAATTAATGTCCATGG Deoxy, MOE, and cEt 51 7879 7894 4083 561281 894 909TTGAATTAATGTCCAT Deoxy, MOE, and cEt 30 7881 7896 4084 561282 896 911TGTTGAATTAATGTCC Deoxy, MOE, and cEt 38 7883 7898 4085 561283 898 913GATGTTGAATTAATGT Deoxy, MOE, and cEt 11 7885 7900 4086 561284 900 915TCGATGTTGAATTAAT Deoxy, MOE, and cEt 20 7887 7902 4087 561285 902 917ATTCGATGTTGAATTA Deoxy, MOE, and cEt 12 7889 7904 4088 561286 904 919CTATTCGATGTTGAAT Deoxy, MOE, and cEt 17 7891 7906 4089 561287 906 921ATCTATTCGATGTTGA Deoxy, MOE, and cEt 32 7893 7908 4090 561288 908 923CCATCTATTCGATGTT Deoxy, MOE, and cEt 69 7895 7910 168 561289 910 925ATCCATCTATTCGATG Deoxy, MOE, and cEt 32 7897 7912 4091 561290 912 927TGATCCATCTATTCGA Deoxy, MOE, and cEt 41 7899 7914 4092 561291 914 929TGTGATCCATCTATTC Deoxy, MOE, and cEt 50 7901 7916 4093 561292 916 931TTTGTGATCCATCTAT Deoxy, MOE, and cEt 50 7903 7918 4094 561293 918 933GTTTTGTGATCCATCT Deoxy, MOE, and cEt 41 7905 7920 4095 561294 920 935AAGTTTTGTGATCCAT Deoxy, MOE, and cEt 56 7907 7922 4096 561295 922 937TGAAGTTTTGTGATCC Deoxy, MOE, and cEt 57 7909 7924 4097 561296 924 939ATTGAAGTTTTGTGAT Deoxy, MOE, and cEt 0 7911 7926 4098 561450 1386 1401CAACATTTTGGTTGAT Deoxy, MOE, and cEt 45 10358 10373 4099 561451 13891404 GATCAACATTTTGGTT Deoxy, MOE, and cEt 33 10361 10376 4100 5614521391 1406 TGGATCAACATTTTGG Deoxy, MOE, and cEt 81 10363 10378 123 5614531393 1408 GATGGATCAACATTTT Deoxy, MOE, and cEt 59 10365 10380 4101561455 1397 1412 GTTGGATGGATCAACA Deoxy, MOE, and cEt 53 10369 103844102 561456 1399 1414 CTGTTGGATGGATCAA Deoxy, MOE, and cEt 71 1037110386 4103 561457 1401 1416 ATCTGTTGGATGGATC Deoxy, MOE, and cEt 7110373 10388 4104 561458 1403 1418 GAATCTGTTGGATGGA Deoxy, MOE, and cEt84 10375 10390 124 561459 1405 1420 CTGAATCTGTTGGATG Deoxy, MOE, and cEt72 10377 10392 4105 561460 1407 1422 TTCTGAATCTGTTGGA Deoxy, MOE, andcEt 78 10379 10394 125 561461 1414 1429 CAAAGCTTTCTGAATC Deoxy, MOE, andcEt 45 10386 10401 4106 561462 1421 1436 GTTCATTCAAAGCTTT Deoxy, MOE,and cEt 87 10393 10408 126 561463 1423 1438 CAGTTCATTCAAAGCT Deoxy, MOE,and cEt 85 10395 10410 127 561464 1425 1440 CTCAGTTCATTCAAAG Deoxy, MOE,and cEt 47 10397 10412 4107 561465 1427 1442 GCCTCAGTTCATTCAA Deoxy,MOE, and cEt 60 10399 10414 4108 561466 1429 1444 TTGCCTCAGTTCATTCDeoxy, MOE, and cEt 68 10401 10416 4109 561467 1431 1446ATTTGCCTCAGTTCAT Deoxy, MOE, and cEt 61 10403 10418 4110 561468 14331448 AAATTTGCCTCAGTTC Deoxy, MOE, and cEt 48 10405 10420 4111 5614691436 1451 TTTAAATTTGCCTCAG Deoxy, MOE, and cEt 59 10408 10423 4112561470 1438 1453 CTTTTAAATTTGCCTC Deoxy, MOE, and cEt 50 10410 104254113 561471 1440 1455 GCCTTTTAAATTTGCC Deoxy, MOE, and cEt 73 1041210427 4114 561472 1452 1467 GTTTAAATTATTGCCT Deoxy, MOE, and cEt 4810424 10439 4115 561473 1463 1478 ATGAGGTTAATGTTTA Deoxy, MOE, and cEt33 10435 10450 4116 561474 1465 1480 GAATGAGGTTAATGTT Deoxy, MOE, andcEt 29 10437 10452 4117 561475 1467 1482 TGGAATGAGGTTAATG Deoxy, MOE,and cEt 66 10439 10454 4118 561476 1469 1484 CTTGGAATGAGGTTAA Deoxy,MOE, and cEt 72 10441 10456 4119 561477 1471 1486 AACTTGGAATGAGGTTDeoxy, MOE, and cEt 69 10443 10458 4120 561478 1473 1488TTAACTTGGAATGAGG Deoxy, MOE, and cEt 74 10445 10460 128 561479 1475 1490CATTAACTTGGAATGA Deoxy, MOE, and cEt 5 10447 10462 4121 561480 1477 1492CACATTAACTTGGAAT Deoxy, MOE, and cEt 26 10449 10464 4122 561481 14791494 ACCACATTAACTTGGA Deoxy, MOE, and cEt 59 10451 10466 4123 5614821481 1496 AGACCACATTAACTTG Deoxy, MOE, and cEt 76 10453 10468 129 5614831483 1498 TTAGACCACATTAACT Deoxy, MOE, and cEt 47 10455 10470 4124561484 1485 1500 TATTAGACCACATTAA Deoxy, MOE, and cEt 38 10457 104724125 561485 1487 1502 ATTATTAGACCACATT Deoxy, MOE, and cEt 59 1045910474 4126 561486 1489 1504 AGATTATTAGACCACA Deoxy, MOE, and cEt 8410461 10476 130 561487 1491 1506 CCAGATTATTAGACCA Deoxy, MOE, and cEt 9310463 10478 131 561488 1493 1508 TACCAGATTATTAGAC Deoxy, MOE, and cEt 2210465 10480 4127 561489 1495 1510 AATACCAGATTATTAG Deoxy, MOE, and cEt48 10467 10482 4128 561490 1497 1512 TTAATACCAGATTATT Deoxy, MOE, andcEt 22 10469 10484 4129 561491 1499 1514 ATTTAATACCAGATTA Deoxy, MOE,and cEt 14 10471 10486 4130 561492 1501 1516 GGATTTAATACCAGAT Deoxy,MOE, and cEt 74 10473 10488 4131 561493 1503 1518 AAGGATTTAATACCAGDeoxy, MOE, and cEt 70 10475 10490 4132 561494 1505 1520TTAAGGATTTAATACC Deoxy, MOE, and cEt 14 10477 10492 4133 561495 15081523 CTCTTAAGGATTTAAT Deoxy, MOE, and cEt 12 10480 10495 4134 5614961510 1525 TTCTCTTAAGGATTTA Deoxy, MOE, and cEt 47 10482 10497 4135561497 1513 1528 GCTTTCTCTTAAGGAT Deoxy, MOE, and cEt 73 10485 105004136 561498 1515 1530 AAGCTTTCTCTTAAGG Deoxy, MOE, and cEt 59 1048710502 4137 561499 1517 1532 TCAAGCTTTCTCTTAA Deoxy, MOE, and cEt 6210489 10504 4138 561500 1526 1541 ATCTATTTCTCAAGCT Deoxy, MOE, and cEt76 10498 10513 132 561501 1547 1562 AGTGACTTTAAGATAA Deoxy, MOE, and cEt23 10519 10534 4139 561502 1549 1564 ACAGTGACTTTAAGAT Deoxy, MOE, andcEt 62 10521 10536 4140 561503 1551 1566 AGACAGTGACTTTAAG Deoxy, MOE,and cEt 55 10523 10538 4141 561504 1553 1568 ATAGACAGTGACTTTA Deoxy,MOE, and cEt 74 10525 10540 133 561505 1555 1570 AAATAGACAGTGACTT Deoxy,MOE, and cEt 59 10527 10542 4142 561506 1557 1572 TTAAATAGACAGTGACDeoxy, MOE, and cEt 38 10529 10544 4143 561507 1559 1574TCTTAAATAGACAGTG Deoxy, MOE, and cEt 54 10531 10546 4144 561508 15611576 AATCTTAAATAGACAG Deoxy, MOE, and cEt 22 10533 10548 4145 5615091563 1578 TTAATCTTAAATAGAC Deoxy, MOE, and cEt 0 10535 10550 4146 5615101565 1580 GTTTAATCTTAAATAG Deoxy, MOE, and cEt 0 10537 10552 4147 5615111569 1584 GTATGTTTAATCTTAA Deoxy, MOE, and cEt 13 10541 10556 4148561512 1572 1587 ATTGTATGTTTAATCT Deoxy, MOE, and cEt 40 10544 105594149 561513 1575 1590 GTGATTGTATGTTTAA Deoxy, MOE, and cEt 71 1054710562 4150 561514 1578 1593 TATGTGATTGTATGTT Deoxy, MOE, and cEt 5810550 10565 4151 561515 1580 1595 GTTATGTGATTGTATG Deoxy, MOE, and cEt68 10552 10567 4152 561516 1582 1597 AGGTTATGTGATTGTA Deoxy, MOE, andcEt 73 10554 10569 4153 561517 1584 1599 TAAGGTTATGTGATTG Deoxy, MOE,and cEt 64 10556 10571 4154 561518 1586 1601 TTTAAGGTTATGTGAT Deoxy,MOE, and cEt 0 10558 10573 4155 561519 1588 1603 TCTTTAAGGTTATGTG Deoxy,MOE, and cEt 53 10560 10575 4156 561520 1590 1605 ATTCTTTAAGGTTATGDeoxy, MOE, and cEt 29 10562 10577 4157 561521 1592 1607GTATTCTTTAAGGTTA Deoxy, MOE, and cEt 24 10564 10579 4158 561522 15941609 CGGTATTCTTTAAGGT Deoxy, MOE, and cEt 70 10566 10581 4159 5615231596 1611 AACGGTATTCTTTAAG Deoxy, MOE, and cEt 42 10568 10583 4160561524 1598 1613 TAAACGGTATTCTTTA Deoxy, MOE, and cEt 26 10570 105854161 561525 1600 1615 TGTAAACGGTATTCTT Deoxy, MOE, and cEt 59 1057210587 4162 561526 1602 1617 AATGTAAACGGTATTC Deoxy, MOE, and cEt 5710574 10589 4142

TABLE 153 Inhibition of ANGPTL3 mRNA by oligonucleotides targeting SEQID NO: 1 and 2 SEQ ID SEQ ID NO: 1 NO: SEQ ID SEQ ID Start 1 Stop % NO:2 NO: 2 SEQ ISIS NO Site Site Sequence Chemistry inhibition Start SiteStop Site ID NO 561681 N/A N/A TCTGGAAGCAGACCTA Deoxy, MOE, and cEt 373096 3111 4164 561682 N/A N/A CTTCTGGAAGCAGACC Deoxy, MOE, and cEt 273098 3113 4165 561683 N/A N/A AAATAAGGTATAGTGA Deoxy, MOE, and cEt 211084 11099 4166 561684 N/A N/A TAGTATTAAGTGTTAA Deoxy, MOE, and cEt 1411133 11148 4167 561685 N/A N/A TCATAGTATTAAGTGT Deoxy, MOE, and cEt 011136 11151 4168 561686 N/A N/A AGATTCCTTTACAATT Deoxy, MOE, and cEt 2111160 11175 4169 561687 N/A N/A ACAAGATTCCTTTACA Deoxy, MOE, and cEt 2111163 11178 4170 561688 N/A N/A CTGACAAGATTCCTTT Deoxy, MOE, and cEt 7011166 11181 4171 561689 N/A N/A AATCTGACAAGATTCC Deoxy, MOE, and cEt 8311169 11184 180 561690 N/A N/A TGTAATCTGACAAGAT Deoxy, MOE, and cEt 4611172 11187 4172 561691 N/A N/A TACTGTAATCTGACAA Deoxy, MOE, and cEt 4711175 11190 4173 561692 N/A N/A TCTTACTGTAATCTGA Deoxy, MOE, and cEt 5011178 11193 4174 561693 N/A N/A CATTCTTACTGTAATC Deoxy, MOE, and cEt 4011181 11196 4175 561694 N/A N/A GTTCATTCTTACTGTA Deoxy, MOE, and cEt 7111184 11199 4176 561695 N/A N/A ATATGTTCATTCTTAC Deoxy, MOE, and cEt 211188 11203 4177 561696 N/A N/A GCCACAAATATGTTCA Deoxy, MOE, and cEt 8011195 11210 4178 561697 N/A N/A GATGCCACAAATATGT Deoxy, MOE, and cEt 7011198 11213 4179 561698 N/A N/A CTCGATGCCACAAATA Deoxy, MOE, and cEt 8011201 11216 181 561699 N/A N/A TAACTCGATGCCACAA Deoxy, MOE, and cEt 8611204 11219 182 561700 N/A N/A CTTTAACTCGATGCCA Deoxy, MOE, and cEt 7711207 11222 4180 561701 N/A N/A AAACTTTAACTCGATG Deoxy, MOE, and cEt 3911210 11225 4181 561702 N/A N/A TATAAACTTTAACTCG Deoxy, MOE, and cEt 1311213 11228 4182 561703 N/A N/A CACAGCATATTTAGGG Deoxy, MOE, and cEt 7111233 11248 4183 561704 N/A N/A TAGAATCACAGCATAT Deoxy, MOE, and cEt 6811239 11254 4184 561705 N/A N/A TATTAGAATCACAGCA Deoxy, MOE, and cEt 7311242 11257 4185 561706 N/A N/A AATGTATTAGAATCAC Deoxy, MOE, and cEt 4011246 11261 4186 561707 N/A N/A ACGAATGTATTAGAAT Deoxy, MOE, and cEt 2211249 11264 4187 561708 N/A N/A TACACGAATGTATTAG Deoxy, MOE, and cEt 3311252 11267 4188 561709 N/A N/A ACCTACACGAATGTAT Deoxy, MOE, and cEt 4211255 11270 4189 561710 N/A N/A AAAACCTACACGAATG Deoxy, MOE, and cEt 2411258 11273 4190 561711 N/A N/A TTGAAAACCTACACGA Deoxy, MOE, and cEt 3411261 11276 4191 561712 N/A N/A TACTTGAAAACCTACA Deoxy, MOE, and cEt 3311264 11279 4192 561713 N/A N/A GTTTATTTCTACTTGA Deoxy, MOE, and cEt 5311273 11288 4193 561714 N/A N/A GAGGTTTATTTCTACT Deoxy, MOE, and cEt 6911276 11291 4194 561715 N/A N/A TACGAGGTTTATTTCT Deoxy, MOE, and cEt 2111279 11294 4195 561716 N/A N/A TGTTACGAGGTTTATT Deoxy, MOE, and cEt 4711282 11297 4196 561717 N/A N/A ACTTGTTACGAGGTTT Deoxy, MOE, and cEt 7011285 11300 4197 561718 N/A N/A CAGTAACTTGTTACGA Deoxy, MOE, and cEt 6011290 11305 4198 561719 N/A N/A GTTCAGTAACTTGTTA Deoxy, MOE, and cEt 4011293 11308 4199 561720 N/A N/A TCAGGCTGTTTAAACG Deoxy, MOE, and cEt 5911308 11323 4200 561721 N/A N/A TTGTCAGGCTGTTTAA Deoxy, MOE, and cEt 7411311 11326 4201 561722 N/A N/A TGCTTGTCAGGCTGTT Deoxy, MOE, and cEt 8211314 11329 183 561723 N/A N/A ACATGCTTGTCAGGCT Deoxy, MOE, and cEt 8411317 11332 184 561724 N/A N/A TATACATGCTTGTCAG Deoxy, MOE, and cEt 7511320 11335 4202 561725 N/A N/A GTCTTTGTTTATTGAA Deoxy, MOE, and cEt 4911347 11362 4203 561726 N/A N/A TGGGTCTTTGTTTATT Deoxy, MOE, and cEt 2711350 11365 4204 561727 N/A N/A GACTGGGTCTTTGTTT Deoxy, MOE, and cEt 2011353 11368 4205 561728 N/A N/A ATAATTTAGGGACTGG Deoxy, MOE, and cEt 2011363 11378 4206 561729 N/A N/A TCTATAATTTAGGGAC Deoxy, MOE, and cEt 3911366 11381 4207 561730 N/A N/A CGATAAACATGCAAGA Deoxy, MOE, and cEt 6811394 11409 4208 561731 N/A N/A TGTCGATAAACATGCA Deoxy, MOE, and cEt 8011397 11412 4209 561732 N/A N/A TGATGTCGATAAACAT Deoxy, MOE, and cEt 6811400 11415 4210 561733 N/A N/A TTGTGATGTCGATAAA Deoxy, MOE, and cEt 2811403 11418 4211 561734 N/A N/A CTGTTGTGATGTCGAT Deoxy, MOE, and cEt 7411406 11421 4212 561735 N/A N/A GATCTGTTGTGATGTC Deoxy, MOE, and cEt 5911409 11424 4213 561736 N/A N/A AGGGATCTGTTGTGAT Deoxy, MOE, and cEt 2411412 11427 4214 561737 N/A N/A TTTAGGGATCTGTTGT Deoxy, MOE, and cEt 1911415 11430 4215 561738 N/A N/A GGATTTAGGGATCTGT Deoxy, MOE, and cEt 2711418 11433 4216 561739 N/A N/A GATTTAGGGATTTAGG Deoxy, MOE, and cEt 4411425 11440 4217 561740 N/A N/A TCTTTAGGGATTTAGG Deoxy, MOE, and cEt 3811433 11448 4218 561741 N/A N/A TAATCTTTAGGGATTT Deoxy, MOE, and cEt 011436 11451 4219 561742 N/A N/A ATCTAATCTTTAGGGA Deoxy, MOE, and cEt 011439 11454 4220 561743 N/A N/A TGTATCTAATCTTTAG Deoxy, MOE, and cEt 1511442 11457 4221 561744 N/A N/A AAATTTGTATCTAATC Deoxy, MOE, and cEt 2111447 11462 4222 561745 N/A N/A GTAAAAAATTTGTATC Deoxy, MOE, and cEt 2311452 11467 4223 561746 N/A N/A GTGGTAAAAAATTTGT Deoxy, MOE, and cEt 3211455 11470 4224 561747 N/A N/A GATACTGTGGTAAAAA Deoxy, MOE, and cEt 4511461 11476 4225 561748 N/A N/A AGTGATACTGTGGTAA Deoxy, MOE, and cEt 6011464 11479 4226 561749 N/A N/A ACAAGTGATACTGTGG Deoxy, MOE, and cEt 7511467 11482 4227 561750 N/A N/A CTGACAAGTGATACTG Deoxy, MOE, and cEt 5911470 11485 4228 561751 N/A N/A ATTCTGACAAGTGATA Deoxy, MOE, and cEt 4811473 11488 4229 561752 N/A N/A TAAATTCTGACAAGTG Deoxy, MOE, and cEt 5911476 11491 4230 561753 N/A N/A TACTGGCAGTTTTAAA Deoxy, MOE, and cEt 4211508 11523 4231 561754 N/A N/A TCTTACTGGCAGTTTT Deoxy, MOE, and cEt 5111511 11526 4232 561755 N/A N/A ATTTCTTACTGGCAGT Deoxy, MOE, and cEt 6911514 11529 4233 561756 N/A N/A AAAATTTCTTACTGGC Deoxy, MOE, and cEt 5711517 11532 4234 561757 N/A N/A AACAAATGGGTTTAAT Deoxy, MOE, and cEt 011535 11550 4235 562374 N/A N/A GAATATTTGCAAGTCT Deoxy, MOE, and cEt 689230 9245 4236 562375 N/A N/A GTAGAGGAATATTTGC Deoxy, MOE, and cEt 839236 9251 151 562376 N/A N/A TCATTGGTAGAGGAAT Deoxy, MOE, and cEt 239242 9257 4237 562377 N/A N/A ATATTTTAAAGTCTCG Deoxy, MOE, and cEt 179258 9273 4238 562378 N/A N/A GTTACATTATTATAGA Deoxy, MOE, and cEt 299273 9288 4239 562379 N/A N/A GTGAAATGTGTTACAT Deoxy, MOE, and cEt 549282 9297 4240 562380 N/A N/A TCACCAGTGAAATGTG Deoxy, MOE, and cEt 649288 9303 4241 562381 N/A N/A CATGTTTCACCAGTGA Deoxy, MOE, and cEt 789294 9309 4242 562382 N/A N/A ACAAGACATGTTTCAC Deoxy, MOE, and cEt 369300 9315 4243 562383 N/A N/A CATATGACAAGACATG Deoxy, MOE, and cEt 429306 9321 4244 562384 N/A N/A CTATAATGCATATGAC Deoxy, MOE, and cEt 59314 9329 4245 562385 N/A N/A TCCTTTCTATAATGCA Deoxy, MOE, and cEt 659320 9335 4246 562386 N/A N/A TGATTATCCTTTCTAT Deoxy, MOE, and cEt 279326 9341 4247 562387 N/A N/A AAAGTCTGATTATCCT Deoxy, MOE, and cEt 909332 9347 152 562388 N/A N/A TAACTGAAAGTCTGAT Deoxy, MOE, and cEt 599338 9353 4248 562389 N/A N/A GTGCACAAAAATGTTA Deoxy, MOE, and cEt 429366 9381 4249 562390 N/A N/A AGCTATGTGCACAAAA Deoxy, MOE, and cEt 779372 9387 4250 562391 N/A N/A GAAGATAGCTATGTGC Deoxy, MOE, and cEt 649378 9393 4251 562392 N/A N/A TTTATTGAAGATAGCT Deoxy, MOE, and cEt 339384 9399 4252 562393 N/A N/A TCATTTTAGTGTATCT Deoxy, MOE, and cEt 409424 9439 4253 562394 N/A N/A CCTTGATCATTTTAGT Deoxy, MOE, and cEt 159430 9445 4254 562395 N/A N/A TGAATCCCTTGATCAT Deoxy, MOE, and cEt 599436 9451 4255 562396 N/A N/A TAGTCTTGAATCCCTT Deoxy, MOE, and cEt 839442 9457 153 562397 N/A N/A GTTGTTTAGTCTTGAA Deoxy, MOE, and cEt 659448 9463 4256 562398 N/A N/A AATTGAGTTGTTTAGT Deoxy, MOE, and cEt 219454 9469 4257 562399 N/A N/A GCAACTAATTGAGTTG Deoxy, MOE, and cEt 159460 9475 4258 562400 N/A N/A ATTGGTGCAACTAATT Deoxy, MOE, and cEt 259466 9481 4259 562401 N/A N/A GTTTTTTATTGGTGCA Deoxy, MOE, and cEt 539473 9488 4260 562402 N/A N/A GGACACTGACAGTTTT Deoxy, MOE, and cEt 439496 9511 4261 562403 N/A N/A CAGGTTGGACACTGAC Deoxy, MOE, and cEt 239502 9517 4262 562404 N/A N/A TAAGTACAGGTTGGAC Deoxy, MOE, and cEt 339508 9523 4263 562405 N/A N/A AGTTATTAAGTACAGG Deoxy, MOE, and cEt 349514 9529 4264 562406 N/A N/A TCTGTGAGTTATTAAG Deoxy, MOE, and cEt 109520 9535 4265 562407 N/A N/A ACCAAAATTCTCCTGA Deoxy, MOE, and cEt 19554 9569 4266 562408 N/A N/A ACCTGAATAACCCTCT Deoxy, MOE, and cEt 739811 9826 4267 562409 N/A N/A GGTATCAGAAAAAGAT Deoxy, MOE, and cEt 149827 9842 4268 562410 N/A N/A AGTATTGGTATCAGAA Deoxy, MOE, and cEt 139833 9848 4269 562411 N/A N/A GGAAGATACTTTGAAG Deoxy, MOE, and cEt 259861 9876 4270 562412 N/A N/A AATGTGGGAAGATACT Deoxy, MOE, and cEt 239867 9882 4271 562413 N/A N/A CAGATAATAGCTAATA Deoxy, MOE, and cEt 299882 9897 4272 562414 N/A N/A TCATTGCAGATAATAG Deoxy, MOE, and cEt 459888 9903 4273 562415 N/A N/A AAGTTGTCATTGCAGA Deoxy, MOE, and cEt 869894 9909 154 562416 N/A N/A GATTCGGATTTTTAAA Deoxy, MOE, and cEt 199909 9924 4274 562417 N/A N/A ATTTGGGATTCGGATT Deoxy, MOE, and cEt 349915 9930 4275 562418 N/A N/A ACGCTTATTTGGGATT Deoxy, MOE, and cEt 649921 9936 4276 562419 N/A N/A TCTAGAGAGAAAACGC Deoxy, MOE, and cEt 649933 9948 4277 562420 N/A N/A AGTTAAGAGGTTTTCG Deoxy, MOE, and cEt 349949 9964 4278 562421 N/A N/A CATTATAGTTAAGAGG Deoxy, MOE, and cEt 249955 9970 4279 562422 N/A N/A CACTTTCATTATAGTT Deoxy, MOE, and cEt 139961 9976 4280 562423 N/A N/A TAGAATGAACACTTTC Deoxy, MOE, and cEt 639970 9985 4281 562424 N/A N/A TTGAACTAGAATGAAC Deoxy, MOE, and cEt 169976 9991 4282 562425 N/A N/A ACCTGATTGAACTAGA Deoxy, MOE, and cEt 519982 9997 4283 562426 N/A N/A TAAAATACCTGATTGA Deoxy, MOE, and cEt 199988 10003 4284 562427 N/A N/A TAGAGGTAAAATACCT Deoxy, MOE, and cEt 129994 10009 4285 562428 N/A N/A GAAGATTAGAGGTAAA Deoxy, MOE, and cEt 110000 10015 4286 562429 N/A N/A TCTGAGGAAGATTAGA Deoxy, MOE, and cEt 3110006 10021 4287 562430 N/A N/A TATACACTACCAAAAA Deoxy, MOE, and cEt 010030 10045 4288 562431 N/A N/A ATAATCTATACACTAC Deoxy, MOE, and cEt 010036 10051 4289 562432 N/A N/A TAAGTCCCAATTTTAA Deoxy, MOE, and cEt 3310065 10080 4290 562433 N/A N/A TCTGTATAAGTCCCAA Deoxy, MOE, and cEt 8910071 10086 155 562434 N/A N/A CCAGTTTTAAATAATC Deoxy, MOE, and cEt 2010085 10100 4291 562435 N/A N/A TGTATCCCAGTTTTAA Deoxy, MOE, and cEt 4410091 10106 4292 562436 N/A N/A GATGCATGTATCCCAG Deoxy, MOE, and cEt 9110097 10112 156 562437 N/A N/A GTTTTAGATGCATGTA Deoxy, MOE, and cEt 6910103 10118 4293 562438 N/A N/A TACAGTGTTTTAGATG Deoxy, MOE, and cEt 2810109 10124 4294 562439 N/A N/A GTAAGTTTATCTTCCT Deoxy, MOE, and cEt 7810138 10153 157 562440 N/A N/A TTCCCCGTAAGTTTAT Deoxy, MOE, and cEt 3310144 10159 4295 562441 N/A N/A CTGTATTTCCCCGTAA Deoxy, MOE, and cEt 5510150 10165 4296 562442 N/A N/A CTGTTACTGTATTTCC Deoxy, MOE, and cEt 7910156 10171 158 562443 N/A N/A TAGTTACTGTTACTGT Deoxy, MOE, and cEt 7010162 10177 4297 562444 N/A N/A CGTATGTAGTTACTGT Deoxy, MOE, and cEt 6610168 10183 4298 562445 N/A N/A AATGGGTACAGACTCG Deoxy, MOE, and cEt 7210182 10197 4299 562446 N/A N/A GCAATTTAATGGGTAC Deoxy, MOE, and cEt 5910189 10204 4300 562447 N/A N/A GATAGATATGCAATTT Deoxy, MOE, and cEt 2010198 10213 4301 562448 N/A N/A AAAGGAGATAGATATG Deoxy, MOE, and cEt 2210204 10219 4302 562449 N/A N/A CCTCCTAAAGGAGATA Deoxy, MOE, and cEt 4210210 10225 4303 562450 N/A N/A CACCAGCCTCCTAAAG Deoxy, MOE, and cEt 3710216 10231 4304 544120 707 726 AGTTCTTGGTGCTCTTGGCT 5-10-5 MOE 83 67206739 15 560990 709 724 TTCTTGGTGCTCTTGG Deoxy, MOE, and cEt 89 6722 6737111 337487 804 823 CACTTGTATGTTCACCTCTG 5-10-5 MOE 81 7389 7408 28561373 1197 1212 TTTGTGATCCCAAGTA Deoxy, MOE, and cEt 40 9772 9787 4305561374 1199 1214 GCTTTGTGATCCCAAG Deoxy, MOE, and cEt 76 9774 9789 4306561375 1201 1216 TTGCTTTGTGATCCCA Deoxy, MOE, and cEt 82 9776 9791 4307561376 1203 1218 TTTTGCTTTGTGATCC Deoxy, MOE, and cEt 40 9778 9793 4308561377 1205 1220 CCTTTTGCTTTGTGAT Deoxy, MOE, and cEt 38 9780 9795 4309561378 1207 1222 GTCCTTTTGCTTTGTG Deoxy, MOE, and cEt 75 9782 9797 4310561379 1209 1224 GTGTCCTTTTGCTTTG Deoxy, MOE, and cEt 40 9784 9799 4311561380 1212 1227 GAAGTGTCCTTTTGCT Deoxy, MOE, and cEt 23 9787 9802 4312561381 1214 1229 TTGAAGTGTCCTTTTG Deoxy, MOE, and cEt 26 9789 9804 4313561382 1216 1231 AGTTGAAGTGTCCTTT Deoxy, MOE, and cEt 34 9791 9806 4314561383 1218 1233 ACAGTTGAAGTGTCCT Deoxy, MOE, and cEt 27 9793 9808 4315561384 1220 1235 GGACAGTTGAAGTGTC Deoxy, MOE, and cEt 19 9795 9810 4316561385 1222 1237 CTGGACAGTTGAAGTG Deoxy, MOE, and cEt 34 9797 9812 4317561386 1224 1239 CTCTGGACAGTTGAAG Deoxy, MOE, and cEt 19 9799 9814 4318561387 1226 1241 CCCTCTGGACAGTTGA Deoxy, MOE, and cEt 54 9801 9816 4319561388 1228 1243 AACCCTCTGGACAGTT Deoxy, MOE, and cEt 50 9803 9818 4320561389 1230 1245 ATAACCCTCTGGACAG Deoxy, MOE, and cEt 35 9805 9820 4321561390 1232 1247 GAATAACCCTCTGGAC Deoxy, MOE, and cEt 34 9807 9822 4322561391 1234 1249 CTGAATAACCCTCTGG Deoxy, MOE, and cEt 62 9809 9824 4323561392 1236 1251 TCCTGAATAACCCTCT Deoxy, MOE, and cEt 57 N/A N/A 4324561393 1238 1253 CCTCCTGAATAACCCT Deoxy, MOE, and cEt 30 N/A N/A 4325561394 1246 1261 ACCACCAGCCTCCTGA Deoxy, MOE, and cEt 70 N/A N/A 4326561395 1251 1266 ATGCCACCACCAGCCT Deoxy, MOE, and cEt 68 10223 102384327 561396 1253 1268 TCATGCCACCACCAGC Deoxy, MOE, and cEt 72 1022510240 4328 561397 1255 1270 CATCATGCCACCACCA Deoxy, MOE, and cEt 6710227 10242 4329 561398 1257 1272 CTCATCATGCCACCAC Deoxy, MOE, and cEt77 10229 10244 172 561399 1259 1274 CACTCATCATGCCACC Deoxy, MOE, and cEt74 10231 10246 2330 561400 1261 1276 CACACTCATCATGCCA Deoxy, MOE, andcEt 80 10233 10248 173 561401 1263 1278 TCCACACTCATCATGC Deoxy, MOE, andcEt 64 10235 10250 4331 561402 1265 1280 TCTCCACACTCATCAT Deoxy, MOE,and cEt 42 10237 10252 4332 561403 1267 1282 TTTCTCCACACTCATC Deoxy,MOE, and cEt 47 10239 10254 4333 561404 1269 1284 GTTTTCTCCACACTCADeoxy, MOE, and cEt 77 10241 10256 4334 561405 1272 1287GTTGTTTTCTCCACAC Deoxy, MOE, and cEt 53 10244 10259 4335 561406 12741289 AGGTTGTTTTCTCCAC Deoxy, MOE, and cEt 67 10246 10261 4336 5614071276 1291 TTAGGTTGTTTTCTCC Deoxy, MOE, and cEt 73 10248 10263 4337561408 1282 1297 TACCATTTAGGTTGTT Deoxy, MOE, and cEt 30 10254 102694338 561409 1284 1299 TTTACCATTTAGGTTG Deoxy, MOE, and cEt 22 1025610271 4339 561410 1286 1301 TATTTACCATTTAGGT Deoxy, MOE, and cEt 2410258 10273 4340 561411 1292 1307 TTGTTATATTTACCAT Deoxy, MOE, and cEt41 10264 10279 4341 561412 1294 1309 GTTTGTTATATTTACC Deoxy, MOE, andcEt 37 10266 10281 4342 561413 1298 1313 CTTGGTTTGTTATATT Deoxy, MOE,and cEt 45 10270 10285 4343 561414 1300 1315 CTCTTGGTTTGTTATA Deoxy,MOE, and cEt 73 10272 10287 4344 561415 1302 1317 TGCTCTTGGTTTGTTADeoxy, MOE, and cEt 45 10274 10289 4345 561416 1304 1319TTTGCTCTTGGTTTGT Deoxy, MOE, and cEt 67 10276 10291 4346 561417 13071322 GATTTTGCTCTTGGTT Deoxy, MOE, and cEt 75 10279 10294 4347 5614181309 1324 TAGATTTTGCTCTTGG Deoxy, MOE, and cEt 87 10281 10296 169 5614191311 1326 CTTAGATTTTGCTCTT Deoxy, MOE, and cEt 64 10283 10298 4348561420 1313 1328 GGCTTAGATTTTGCTC Deoxy, MOE, and cEt 58 10285 103004349 561421 1315 1330 CTGGCTTAGATTTTGC Deoxy, MOE, and cEt 70 1028710302 4350 561422 1317 1332 CTCTGGCTTAGATTTT Deoxy, MOE, and cEt 3810289 10304 4351 561423 1319 1334 CTCTCTGGCTTAGATT Deoxy, MOE, and cEt63 10291 10306 4352 561424 1321 1336 TCCTCTCTGGCTTAGA Deoxy, MOE, andcEt 76 10293 10308 4353 561425 1323 1338 TCTCCTCTCTGGCTTA Deoxy, MOE,and cEt 67 10295 10310 4354 561426 1332 1347 TAATCCTCTTCTCCTC Deoxy,MOE, and cEt 50 10304 10319 4355 561427 1334 1349 GATAATCCTCTTCTCCDeoxy, MOE, and cEt 36 10306 10321 4356 561428 1336 1351AAGATAATCCTCTTCT Deoxy, MOE, and cEt 43 10308 10323 4357 561429 13381353 CCAAGATAATCCTCTT Deoxy, MOE, and cEt 59 10310 10325 4358 5614301340 1355 TTCCAAGATAATCCTC Deoxy, MOE, and cEt 65 10312 10327 4359561431 1342 1357 ACTTCCAAGATAATCC Deoxy, MOE, and cEt 74 10314 103294360 561432 1344 1359 AGACTTCCAAGATAAT Deoxy, MOE, and cEt 52 1031610331 4361 561433 1346 1361 TGAGACTTCCAAGATA Deoxy, MOE, and cEt 4910318 10333 4362 561434 1348 1363 TTTGAGACTTCCAAGA Deoxy, MOE, and cEt47 10320 10335 4363 561435 1350 1365 ATTTTGAGACTTCCAA Deoxy, MOE, andcEt 64 10322 10337 4364 561436 1352 1367 CCATTTTGAGACTTCC Deoxy, MOE,and cEt 84 10324 10339 170 561437 1354 1369 TTCCATTTTGAGACTT Deoxy, MOE,and cEt 67 10326 10341 4365 561438 1356 1371 CCTTCCATTTTGAGAC Deoxy,MOE, and cEt 53 10328 10343 4366 561439 1358 1373 AACCTTCCATTTTGAGDeoxy, MOE, and cEt 37 10330 10345 4367 561440 1360 1375ATAACCTTCCATTTTG Deoxy, MOE, and cEt 50 10332 10347 4368 561441 13621377 GTATAACCTTCCATTT Deoxy, MOE, and cEt 27 10334 10349 4369 5614421364 1379 GAGTATAACCTTCCAT Deoxy, MOE, and cEt 65 10336 10351 4370561443 1366 1381 TAGAGTATAACCTTCC Deoxy, MOE, and cEt 84 10338 10353 171561444 1368 1383 TATAGAGTATAACCTT Deoxy, MOE, and cEt 17 10340 103554371 561445 1370 1385 TTTATAGAGTATAACC Deoxy, MOE, and cEt 37 1034210357 4372 561446 1373 1388 GATTTTATAGAGTATA Deoxy, MOE, and cEt 2810345 10360 4373 561447 1375 1390 TTGATTTTATAGAGTA Deoxy, MOE, and cEt21 10347 10362 4374 561448 1377 1392 GGTTGATTTTATAGAG Deoxy, MOE, andcEt 28 10349 10364 4375 561449 1379 1394 TTGGTTGATTTTATAG Deoxy, MOE,and cEt 22 10351 10366 4376 567295 1452 1471 TAATGTTTAAATTATTGCCT 5-10-5MOE 43 10424 10443 4377 567296 1455 1474 GGTTAATGTTTAAATTATTG 5-10-5 MOE22 10427 10446 4378 567297 1456 1475 AGGTTAATGTTTAAATTATT 5-10-5 MOE 010428 10447 4379 567298 1457 1476 GAGGTTAATGTTTAAATTAT 5-10-5 MOE 010429 10448 4380 567299 1458 1477 TGAGGTTAATGTTTAAATTA 5-10-5 MOE 610430 10449 4381 567300 1460 1479 AATGAGGTTAATGTTTAAAT 5-10-5 MOE 1410432 10451 4382 567301 1461 1480 GAATGAGGTTAATGTTTAAA 5-10-5 MOE 510433 10452 4383 567302 1462 1481 GGAATGAGGTTAATGTTTAA 5-10-5 MOE 2710434 10453 4384 567303 1463 1482 TGGAATGAGGTTAATGTTTA 5-10-5 MOE 3210435 10454 4385 567304 1464 1483 TTGGAATGAGGTTAATGTTT 5-10-5 MOE 3710436 10455 4386 567305 1465 1484 CTTGGAATGAGGTTAATGTT 5-10-5 MOE 2510437 10456 4387 567306 1468 1487 TAACTTGGAATGAGGTTAAT 5-10-5 MOE 2910440 10459 4388 567307 1469 1488 TTAACTTGGAATGAGGTTAA 5-10-5 MOE 4410441 10460 4389 337513 1470 1489 ATTAACTTGGAATGAGGTTA 5-10-5 MOE 5210442 10461 4390 567308 1471 1490 CATTAACTTGGAATGAGGTT 5-10-5 MOE 6210443 10462 4391 567309 1472 1491 ACATTAACTTGGAATGAGGT 5-10-5 MOE 5810444 10463 4392 567310 1473 1492 CACATTAACTTGGAATGAGG 5-10-5 MOE 7810445 10464 92 567311 1475 1494 ACCACATTAACTTGGAATGA 5-10-5 MOE 59 1044710466 4393 567312 1476 1495 GACCACATTAACTTGGAATG 5-10-5 MOE 57 1044810467 4394 337514 1477 1496 AGACCACATTAACTTGGAAT 5-10-5 MOE 71 1044910468 4395 567313 1478 1497 TAGACCACATTAACTTGGAA 5-10-5 MOE 43 1045010469 4396 567314 1479 1498 TTAGACCACATTAACTTGGA 5-10-5 MOE 59 1045110470 4397 567315 1480 1499 ATTAGACCACATTAACTTGG 5-10-5 MOE 70 1045210471 4398 567316 1481 1500 TATTAGACCACATTAACTTG 5-10-5 MOE 53 1045310472 4399 567317 1482 1501 TTATTAGACCACATTAACTT 5-10-5 MOE 49 1045410473 4400 567318 1483 1502 ATTATTAGACCACATTAACT 5-10-5 MOE 41 1045510474 4401 567319 1484 1503 GATTATTAGACCACATTAAC 5-10-5 MOE 47 1045610475 4402 567320 1487 1506 CCAGATTATTAGACCACATT 5-10-5 MOE 86 1045910478 93 567321 1489 1508 TACCAGATTATTAGACCACA 5-10-5 MOE 85 10461 1048094 337516 1490 1509 ATACCAGATTATTAGACCAC 5-10-5 MOE 77 10462 10481 86567322 1491 1510 AATACCAGATTATTAGACCA 5-10-5 MOE 50 10463 10482 4403567323 1492 1511 TAATACCAGATTATTAGACC 5-10-5 MOE 56 10464 10483 4404567324 1494 1513 TTTAATACCAGATTATTAGA 5-10-5 MOE 9 10466 10485 4405567325 1495 1514 ATTTAATACCAGATTATTAG 5-10-5 MOE 24 10467 10486 4406567326 1496 1515 GATTTAATACCAGATTATTA 5-10-5 MOE 37 10468 10487 4407567327 1500 1519 TAAGGATTTAATACCAGATT 5-10-5 MOE 60 10472 10491 4408567328 1507 1526 TTTCTCTTAAGGATTTAATA 5-10-5 MOE 34 10479 10498 4409567329 1508 1527 CTTTCTCTTAAGGATTTAAT 5-10-5 MOE 46 10480 10499 4410567330 1509 1528 GCTTTCTCTTAAGGATTTAA 5-10-5 MOE 75 10481 10500 95567331 1510 1529 AGCTTTCTCTTAAGGATTTA 5-10-5 MOE 59 10482 10501 4411567332 1511 1530 AAGCTTTCTCTTAAGGATTT 5-10-5 MOE 30 10483 10502 4412567333 1513 1532 TCAAGCTTTCTCTTAAGGAT 5-10-5 MOE 65 10485 10504 4413567334 1514 1533 CTCAAGCTTTCTCTTAAGGA 5-10-5 MOE 77 10486 10505 96567335 1515 1534 TCTCAAGCTTTCTCTTAAGG 5-10-5 MOE 75 10487 10506 97567336 1516 1535 TTCTCAAGCTTTCTCTTAAG 5-10-5 MOE 59 10488 10507 4414567337 1517 1536 TTTCTCAAGCTTTCTCTTAA 5-10-5 MOE 52 10489 10508 4415567338 1521 1540 TCTATTTCTCAAGCTTTCTC 5-10-5 MOE 68 10493 10512 4416567339 1522 1541 ATCTATTTCTCAAGCTTTCT 5-10-5 MOE 71 10494 10513 4417567340 1523 1542 AATCTATTTCTCAAGCTTTC 5-10-5 MOE 74 10495 10514 4418567341 1524 1543 AAATCTATTTCTCAAGCTTT 5-10-5 MOE 63 10496 10515 4419567342 1525 1544 AAAATCTATTTCTCAAGCTT 5-10-5 MOE 54 10497 10516 4420567343 1532 1551 GATAAAAAAAATCTATTTCT 5-10-5 MOE 30 10504 10523 4421567344 1548 1567 TAGACAGTGACTTTAAGATA 5-10-5 MOE 37 10520 10539 4422567345 1549 1568 ATAGACAGTGACTTTAAGAT 5-10-5 MOE 29 10521 10540 4423567346 1550 1569 AATAGACAGTGACTTTAAGA 5-10-5 MOE 48 10522 10541 4424567347 1551 1570 AAATAGACAGTGACTTTAAG 5-10-5 MOE 26 10523 10542 4425567348 1552 1571 TAAATAGACAGTGACTTTAA 5-10-5 MOE 26 10524 10543 4426567349 1553 1572 TTAAATAGACAGTGACTTTA 5-10-5 MOE 50 10525 10544 4427567350 1554 1573 CTTAAATAGACAGTGACTTT 5-10-5 MOE 63 10526 10545 4428567351 1555 1574 TCTTAAATAGACAGTGACTT 5-10-5 MOE 57 10527 10546 4429567352 1556 1575 ATCTTAAATAGACAGTGACT 5-10-5 MOE 69 10528 10547 4430567353 1557 1576 AATCTTAAATAGACAGTGAC 5-10-5 MOE 40 10529 10548 4431567354 1558 1577 TAATCTTAAATAGACAGTGA 5-10-5 MOE 30 10530 10549 4432567355 1559 1578 TTAATCTTAAATAGACAGTG 5-10-5 MOE 25 10531 10550 4433567356 1560 1579 TTTAATCTTAAATAGACAGT 5-10-5 MOE 0 10532 10551 4434567357 1561 1580 GTTTAATCTTAAATAGACAG 5-10-5 MOE 34 10533 10552 4435567358 1562 1581 TGTTTAATCTTAAATAGACA 5-10-5 MOE 5 10534 10553 4436567359 1563 1582 ATGTTTAATCTTAAATAGAC 5-10-5 MOE 0 10535 10554 4437567360 1567 1586 TTGTATGTTTAATCTTAAAT 5-10-5 MOE 0 10539 10558 4438567361 1568 1587 ATTGTATGTTTAATCTTAAA 5-10-5 MOE 8 10540 10559 4439567362 1569 1588 GATTGTATGTTTAATCTTAA 5-10-5 MOE 20 10541 10560 4440567363 1570 1589 TGATTGTATGTTTAATCTTA 5-10-5 MOE 29 10542 10561 4441567364 1574 1593 TATGTGATTGTATGTTTAAT 5-10-5 MOE 7 10546 10565 4442567365 1576 1595 GTTATGTGATTGTATGTTTA 5-10-5 MOE 43 10548 10567 4443567366 1580 1599 TAAGGTTATGTGATTGTATG 5-10-5 MOE 28 10552 10571 4444567367 1581 1600 TTAAGGTTATGTGATTGTAT 5-10-5 MOE 31 10553 10572 4445567368 1585 1604 TTCTTTAAGGTTATGTGATT 5-10-5 MOE 12 10557 10576 4446561527 1604 1619 GAAATGTAAACGGTAT Deoxy, MOE, and cEt 47 10576 105914447 561528 1606 1621 GAGAAATGTAAACGGT Deoxy, MOE, and cEt 89 1057810593 174 561529 1608 1623 TTGAGAAATGTAAACG Deoxy, MOE, and cEt 55 1058010595 4448 561530 1611 1626 TGATTGAGAAATGTAA Deoxy, MOE, and cEt 1810583 10598 4449 561531 1613 1628 TTTGATTGAGAAATGT Deoxy, MOE, and cEt30 10585 10600 4450 561532 1619 1634 AAGAATTTTGATTGAG Deoxy, MOE, andcEt 53 10591 10606 4451 561533 1621 1636 ATAAGAATTTTGATTG Deoxy, MOE,and cEt 29 10593 10608 4452 561534 1632 1647 CAAATAGTATTATAAG Deoxy,MOE, and cEt 6 10604 10619 4453 561535 1653 1668 CCCACATCACAAAATT Deoxy,MOE, and cEt 70 10625 10640 4454 561536 1657 1672 GATTCCCACATCACAADeoxy, MOE, and cEt 77 10629 10644 4455 561537 1659 1674TTGATTCCCACATCAC Deoxy, MOE, and cEt 78 10631 10646 4456 561538 16611676 AATTGATTCCCACATC Deoxy, MOE, and cEt 68 10633 10648 4457 5615391663 1678 AAAATTGATTCCCACA Deoxy, MOE, and cEt 72 10635 10650 4458561540 1665 1680 CTAAAATTGATTCCCA Deoxy, MOE, and cEt 54 10637 106524459 561541 1668 1683 CATCTAAAATTGATTC Deoxy, MOE, and cEt 0 10640 106554460 561542 1670 1685 ACCATCTAAAATTGAT Deoxy, MOE, and cEt 35 1064210657 4461 561543 1672 1687 TGACCATCTAAAATTG Deoxy, MOE, and cEt 5510644 10659 4462 561544 1674 1689 TGTGACCATCTAAAAT Deoxy, MOE, and cEt56 10646 10661 4463 561545 1676 1691 ATTGTGACCATCTAAA Deoxy, MOE, andcEt 73 10648 10663 4464 561546 1678 1693 AGATTGTGACCATCTA Deoxy, MOE,and cEt 67 10650 10665 4465 561547 1680 1695 CTAGATTGTGACCATC Deoxy,MOE, and cEt 50 10652 10667 4466 561548 1682 1697 ATCTAGATTGTGACCADeoxy, MOE, and cEt 77 10654 10669 4467 561549 1684 1699TAATCTAGATTGTGAC Deoxy, MOE, and cEt 55 10656 10671 4468 561550 16861701 TATAATCTAGATTGTG Deoxy, MOE, and cEt 28 10658 10673 4469 5615511688 1703 ATTATAATCTAGATTG Deoxy, MOE, and cEt 52 10660 10675 4470561552 1690 1705 TGATTATAATCTAGAT Deoxy, MOE, and cEt 43 10662 106774471 561553 1692 1707 ATTGATTATAATCTAG Deoxy, MOE, and cEt 53 1066410679 4472 561554 1694 1709 CTATTGATTATAATCT Deoxy, MOE, and cEt 5410666 10681 4473 561555 1696 1711 ACCTATTGATTATAAT Deoxy, MOE, and cEt44 10668 10683 4474 561556 1698 1713 TCACCTATTGATTATA Deoxy, MOE, andcEt 52 10670 10685 4475 561557 1700 1715 GTTCACCTATTGATTA Deoxy, MOE,and cEt 50 10672 10687 4476 561558 1702 1717 AAGTTCACCTATTGAT Deoxy,MOE, and cEt 58 10674 10689 4477 561559 1704 1719 ATAAGTTCACCTATTGDeoxy, MOE, and cEt 66 10676 10691 4478 561560 1706 1721TAATAAGTTCACCTAT Deoxy, MOE, and cEt 38 10678 10693 4479 561561 17081723 TTTAATAAGTTCACCT Deoxy, MOE, and cEt 50 10680 10695 4480 5615621710 1725 TATTTAATAAGTTCAC Deoxy, MOE, and cEt 32 10682 10697 4481561563 1712 1727 GTTATTTAATAAGTTC Deoxy, MOE, and cEt 47 10684 106994482 561564 1761 1776 CATATGATGCCTTTTA Deoxy, MOE, and cEt 63 1073310748 4483 561565 1763 1778 CTCATATGATGCCTTT Deoxy, MOE, and cEt 8110735 10750 175 561566 1765 1780 AGCTCATATGATGCCT Deoxy, MOE, and cEt 8110737 10752 176 561567 1767 1782 TTAGCTCATATGATGC Deoxy, MOE, and cEt 8410739 10754 177 561568 1769 1784 TATTAGCTCATATGAT Deoxy, MOE, and cEt 4610741 10756 4484 561569 1771 1786 GATATTAGCTCATATG Deoxy, MOE, and cEt49 10743 10758 4485 561570 1773 1788 GTGATATTAGCTCATA Deoxy, MOE, andcEt 81 10745 10760 4486 561571 1775 1790 TTGTGATATTAGCTCA Deoxy, MOE,and cEt 85 10747 10762 178 561572 1777 1792 AGTTGTGATATTAGCT Deoxy, MOE,and cEt 68 10749 10764 4487 561573 1779 1794 AAAGTTGTGATATTAG Deoxy,MOE, and cEt 45 10751 10766 4488 561574 1781 1796 GGAAAGTTGTGATATTDeoxy, MOE, and cEt 27 10753 10768 4489 561575 1783 1798TGGGAAAGTTGTGATA Deoxy, MOE, and cEt 36 10755 10770 4490 561576 17851800 ACTGGGAAAGTTGTGA Deoxy, MOE, and cEt 83 10757 10772 179 561577 17871802 AAACTGGGAAAGTTGT Deoxy, MOE, and cEt 56 10759 10774 4491 5615781789 1804 TTAAACTGGGAAAGTT Deoxy, MOE, and cEt 44 10761 10776 4492561579 1794 1809 GTTTTTTAAACTGGGA Deoxy, MOE, and cEt 58 10766 107814493 561580 1796 1811 TAGTTTTTTAAACTGG Deoxy, MOE, and cEt 0 10768 107834494 561581 1802 1817 GAGTACTAGTTTTTTA Deoxy, MOE, and cEt 18 1077410789 4495 561582 1804 1819 AAGAGTACTAGTTTTT Deoxy, MOE, and cEt 5510776 10791 4496 561583 1806 1821 ACAAGAGTACTAGTTT Deoxy, MOE, and cEt51 10778 10793 4497 561584 1808 1823 TAACAAGAGTACTAGT Deoxy, MOE, andcEt 53 10780 10795 4498 561585 1810 1825 TTTAACAAGAGTACTA Deoxy, MOE,and cEt 48 10782 10797 4499 561586 1812 1827 GTTTTAACAAGAGTAC Deoxy,MOE, and cEt 49 10784 10799 4500 561587 1814 1829 GAGTTTTAACAAGAGTDeoxy, MOE, and cEt 54 10786 10801 4501 561588 1816 1831TAGAGTTTTAACAAGA Deoxy, MOE, and cEt 9 10788 10803 4502 561589 1819 1834GTTTAGAGTTTTAACA Deoxy, MOE, and cEt 24 10791 10806 4503 561590 18221837 CAAGTTTAGAGTTTTA Deoxy, MOE, and cEt 30 10794 10809 4504 5615911824 1839 GTCAAGTTTAGAGTTT Deoxy, MOE, and cEt 60 10796 10811 4505561592 1826 1841 TAGTCAAGTTTAGAGT Deoxy, MOE, and cEt 56 10798 108134506 561593 1828 1843 TTTAGTCAAGTTTAGA Deoxy, MOE, and cEt 41 1080010815 4507 561594 1830 1845 TATTTAGTCAAGTTTA Deoxy, MOE, and cEt 1410802 10817 4508 561595 1832 1847 TGTATTTAGTCAAGTT Deoxy, MOE, and cEt39 10804 10819 4509 561596 1834 1849 TCTGTATTTAGTCAAG Deoxy, MOE, andcEt 51 10806 10821 4510 561597 1836 1851 CCTCTGTATTTAGTCA Deoxy, MOE,and cEt 72 10808 10823 4511 561598 1838 1853 GTCCTCTGTATTTAGT Deoxy,MOE, and cEt 55 10810 10825 4512 561599 1840 1855 CAGTCCTCTGTATTTADeoxy, MOE, and cEt 63 10812 10827 4513 561600 1842 1857ACCAGTCCTCTGTATT Deoxy, MOE, and cEt 66 10814 10829 4514 561601 18441859 TTACCAGTCCTCTGTA Deoxy, MOE, and cEt 57 10816 10831 4515 5616021846 1861 AATTACCAGTCCTCTG Deoxy, MOE, and cEt 43 10818 10833 4516561603 1848 1863 ACAATTACCAGTCCTC Deoxy, MOE, and cEt 67 10820 108354517

TABLE 154 Inhibition of ANGPTL3 mRNA by oligonucleotides targeting SEQID NO: 1 and 2 SEQ SEQ ID SEQ ID SEQ ID NO: 1 NO: ID NO: NO: 2 SEQ ISISStart 1 Stop % 2 Start Stop ID NO Site Site Sequence Chemistryinhibition Site Site NO 561835 N/A N/A GCAAATTTTCAGTGTT Deoxy, MOE, andcEt 49 3850 3865 4518 561836 N/A N/A CGATTTGTAATTTTCA Deoxy, MOE, andcEt 20 3874 3889 4519 561837 N/A N/A TTTAACCGATTTGTAA Deoxy, MOE, andcEt 42 3880 3895 4520 561838 N/A N/A GTATAATTTAACCGAT Deoxy, MOE, andcEt 15 3886 3901 4521 561839 N/A N/A CTAGATTGTATAATTT Deoxy, MOE, andcEt 15 3893 3908 4522 561840 N/A N/A AGTGTTCTAGATTGTA Deoxy, MOE, andcEt 45 3899 3914 4523 561841 N/A N/A TGACATAGTGTTCTAG Deoxy, MOE, andcEt 51 3905 3920 4524 561842 N/A N/A GTGTAATGACATAGTG Deoxy, MOE, andcEt 58 3911 3926 4525 561843 N/A N/A ACAATAGTGTAATGAC Deoxy, MOE, andcEt 12 3917 3932 4526 561844 N/A N/A GTAATTTACAATAGTG Deoxy, MOE, andcEt 18 3924 3939 4527 561845 N/A N/A CCTTCAGTAATTTACA Deoxy, MOE, andcEt 0 3930 3945 4528 561846 N/A N/A TACTTACCTTCAGTAA Deoxy, MOE, and cEt2 3936 3951 4529 561847 N/A N/A CTGGAGAATAGTTTTA Deoxy, MOE, and cEt 193969 3984 4530 561848 N/A N/A TTAAACACTGGAGAAT Deoxy, MOE, and cEt 143976 3991 4531 561849 N/A N/A GCCCAGCATATTTTCA Deoxy, MOE, and cEt 224034 4049 4532 561850 N/A N/A GAAAAAGCCCAGCATA Deoxy, MOE, and cEt 154040 4055 4533 561851 N/A N/A GATTTTCTGAACTTCA Deoxy, MOE, and cEt 524063 4078 4534 561852 N/A N/A GTACTATCTCTAAAAT Deoxy, MOE, and cEt 64081 4096 4535 561853 N/A N/A TAAATTGTACTATCTC Deoxy, MOE, and cEt 134087 4102 4536 561854 N/A N/A CACATATTTTTGTCCT Deoxy, MOE, and cEt 474115 4130 4537 561855 N/A N/A CTTTCAAATAGCACAT Deoxy, MOE, and cEt 314126 4141 4538 561856 N/A N/A GTATGCTTCTTTCAAA Deoxy, MOE, and cEt 224134 4149 4539 561857 N/A N/A CCCCTTGTATGCTTCT Deoxy, MOE, and cEt 554140 4155 4540 561858 N/A N/A TTCCTTCCCCTTGTAT Deoxy, MOE, and cEt 324146 4161 4541 561859 N/A N/A TGGCAATTCCTTCCCC Deoxy, MOE, and cEt 434152 4167 4542 561860 N/A N/A GAATATTGGCAATTCC Deoxy, MOE, and cEt 524158 4173 4543 561861 N/A N/A CTAATAATGGATTTGA Deoxy, MOE, and cEt 04179 4194 4544 561862 N/A N/A CTATCATAATCTAAAT Deoxy, MOE, and cEt 04202 4217 4545 561863 N/A N/A GTAACACTATCATAAT Deoxy, MOE, and cEt 74208 4223 4546 561864 N/A N/A AATTTCCTGTAACACT Deoxy, MOE, and cEt 174216 4231 4547 561865 N/A N/A AAGTTGCTTTCCTCTT Deoxy, MOE, and cEt 124243 4258 4548 561866 N/A N/A GGTTATAAGTTGCTTT Deoxy, MOE, and cEt 64249 4264 4549 561867 N/A N/A TAGGTTGGTTATAAGT Deoxy, MOE, and cEt 104255 4270 4550 561868 N/A N/A AGAGAGTAGGTTGGTT Deoxy, MOE, and cEt 104261 4276 4551 561869 N/A N/A GGATATAGAGAGTAGG Deoxy, MOE, and cEt 234267 4282 4552 561870 N/A N/A AAGTCTGGATATAGAG Deoxy, MOE, and cEt 134273 4288 4553 561871 N/A N/A CTACAAAAGTCTGGAT Deoxy, MOE, and cEt 14279 4294 4554 561872 N/A N/A CTTACCTGATTTTCTA Deoxy, MOE, and cEt 04385 4400 4555 561873 N/A N/A TACTGACTTACCTGAT Deoxy, MOE, and cEt 24391 4406 4556 561874 N/A N/A CCATTAAAATACTGAC Deoxy, MOE, and cEt 14400 4415 4557 561875 N/A N/A GGACATACCATTAAAA Deoxy, MOE, and cEt 114407 4422 4558 561876 N/A N/A AAGATGGGACATACCA Deoxy, MOE, and cEt 384413 4428 4559 561877 N/A N/A GTGTGAAAGATGGGAC Deoxy, MOE, and cEt 254419 4434 4560 561878 N/A N/A AGACCTGTGTGAAAGA Deoxy, MOE, and cEt 334425 4440 4561 561879 N/A N/A TTTTACAGACCTGTGT Deoxy, MOE, and cEt 294431 4446 4562 561880 N/A N/A CAGTGTTTTTACAGAC Deoxy, MOE, and cEt 404437 4452 4563 561881 N/A N/A TAGGATTCAGTGTTTT Deoxy, MOE, and cEt 624444 4459 4564 561882 N/A N/A GTTAAAGCTTGTAAAT Deoxy, MOE, and cEt 164465 4480 4565 561883 N/A N/A GATCCAGTTAAAGCTT Deoxy, MOE, and cEt 394471 4486 4566 561884 N/A N/A ACTCATGATCCAGTTA Deoxy, MOE, and cEt 604477 4492 4567 561885 N/A N/A AATTTTACTCATGATC Deoxy, MOE, and cEt 364483 4498 4568 561886 N/A N/A TGTGATAATTTTACTC Deoxy, MOE, and cEt 304489 4504 4569 561887 N/A N/A TGCTGATGTGATAATT Deoxy, MOE, and cEt 414495 4510 4570 561888 N/A N/A CAGTTATGCTGATGTG Deoxy, MOE, and cEt 864501 4516 185 561889 N/A N/A GCAATTTTAACAGTTA Deoxy, MOE, and cEt 134511 4526 4571 561890 N/A N/A GAGCCTGCAATTTTAA Deoxy, MOE, and cEt 144517 4532 4572 561891 N/A N/A TAGCTTCAGAGCCTGC Deoxy, MOE, and cEt 614525 4540 4573 561892 N/A N/A GTTTATTAGCTTCAGA Deoxy, MOE, and cEt 454531 4546 4574 561893 N/A N/A CAGGTAGTTTATTAGC Deoxy, MOE, and cEt 374537 4552 4575 561894 N/A N/A TAAATGCAGGTAGTTT Deoxy, MOE, and cEt 114543 4558 4576 561895 N/A N/A ATGGTTTAAATGCAGG Deoxy, MOE, and cEt 534549 4564 4577 561896 N/A N/A TAGAGCCATGGTTTAA Deoxy, MOE, and cEt 584556 4571 4578 561897 N/A N/A AAGTTTTAGAGCCATG Deoxy, MOE, and cEt 814562 4577 186 561898 N/A N/A TCACACAAAGTTTTAG Deoxy, MOE, and cEt 174569 4584 4579 561899 N/A N/A GTGAAGTAATTTATTC Deoxy, MOE, and cEt 84589 4604 4580 561900 N/A N/A ACTGAGAGATAAAGGG Deoxy, MOE, and cEt 344605 4620 4581 561901 N/A N/A GTATATGTGAGGAAAC Deoxy, MOE, and cEt 184619 4634 4582 561902 N/A N/A TTTGTAGTATATGTGA Deoxy, MOE, and cEt 34625 4640 4583 561903 N/A N/A ATTATCTTTGTAGTAT Deoxy, MOE, and cEt 84631 4646 4584 561904 N/A N/A ATAAGTTCTGTTATTA Deoxy, MOE, and cEt 184643 4658 4585 561905 N/A N/A AATCCTATAAGTTCTG Deoxy, MOE, and cEt 554649 4664 4586 561906 N/A N/A CTGCTATGAATTAATT Deoxy, MOE, and cEt 164679 4694 4587 561907 N/A N/A CATTGGCTGCTATGAA Deoxy, MOE, and cEt 484685 4700 4588 561908 N/A N/A AGATGACATTGGCTGC Deoxy, MOE, and cEt 714691 4706 4589 561909 N/A N/A TTAGTAAGATGACATT Deoxy, MOE, and cEt 04697 4712 4590 561910 N/A N/A GATCTAATTTGAATTT Deoxy, MOE, and cEt 74712 4727 4591 561911 N/A N/A TTGAGCAAAGAGAAAC Deoxy, MOE, and cEt 64730 4745 4592 561989 N/A N/A GAATGTTGACCTTTAA Deoxy, MOE, and cEt 495356 5371 4593 561990 N/A N/A ATTGTTGAATGTTGAC Deoxy, MOE, and cEt 575362 5377 4594 561991 N/A N/A TTAATTACATTGTTGA Deoxy, MOE, and cEt 05370 5385 4595 561992 N/A N/A TTGTAGATTAATTACA Deoxy, MOE, and cEt 185377 5392 4596 561993 N/A N/A TTTACATTGTAGATTA Deoxy, MOE, and cEt 35383 5398 4597 561994 N/A N/A CAGATGTTTACATTGT Deoxy, MOE, and cEt 715389 5404 4598 561995 N/A N/A CTTCACCAGATGTTTA Deoxy, MOE, and cEt 195395 5410 4599 561996 N/A N/A CTGTCACTTCACCAGA Deoxy, MOE, and cEt 775401 5416 187 561997 N/A N/A AGTGCTTCCCTCTGTC Deoxy, MOE, and cEt 665412 5427 4600 561998 N/A N/A TAAACAAGTGCTTCCC Deoxy, MOE, and cEt 625418 5433 4601 561999 N/A N/A TAGCTTTTTTCTAAAC Deoxy, MOE, and cEt 05429 5444 4602 562000 N/A N/A CTGACATAGCTTTTTT Deoxy, MOE, and cEt 665435 5450 4603 562001 N/A N/A TGGATTCTGACATAGC Deoxy, MOE, and cEt 855441 5456 188 562002 N/A N/A AATACATGGATTCTGA Deoxy, MOE, and cEt 355447 5462 4604 562003 N/A N/A TATTAGAATACATGGA Deoxy, MOE, and cEt 75453 5468 4605 562004 N/A N/A GTACTGCATATTAGAA Deoxy, MOE, and cEt 485461 5476 4606 562005 N/A N/A ACTATTGTACTGCATA Deoxy, MOE, and cEt 535467 5482 4607 562006 N/A N/A TTTTAAACTATTGTAC Deoxy, MOE, and cEt 05473 5488 4608 562007 N/A N/A GAGAGTATTATTAATA Deoxy, MOE, and cEt 85490 5505 4609 562008 N/A N/A CTGTTTGAGAGTATTA Deoxy, MOE, and cEt 05496 5511 4610 562009 N/A N/A GAATAGCTGTTTGAGA Deoxy, MOE, and cEt 345502 5517 4611 562010 N/A N/A AATCCTCTTGAATAGC Deoxy, MOE, and cEt 625511 5526 4612 562011 N/A N/A TTTTTGAATCCTCTTG Deoxy, MOE, and cEt 505517 5532 4613 562012 N/A N/A GAGTTTATATTATGTT Deoxy, MOE, and cEt 55532 5547 4614 562013 N/A N/A GTTTCTCTGAGTTTAT Deoxy, MOE, and cEt 585540 5555 4615 562014 N/A N/A TTACCAGTTTCTCTGA Deoxy, MOE, and cEt 645546 5561 4616 562015 N/A N/A GATTTTGTTTACCAGT Deoxy, MOE, and cEt 685554 5569 4617 562016 N/A N/A GTTTTATATCTCTTGA Deoxy, MOE, and cEt 335574 5589 4618 562017 N/A N/A TTGGTAATAATATTTG Deoxy, MOE, and cEt 135589 5604 4619 562018 N/A N/A TGGAAATTGGTAATAA Deoxy, MOE, and cEt 15595 5610 4620 562019 N/A N/A GTTTAGTGGAAATTGG Deoxy, MOE, and cEt 445601 5616 4621 562020 N/A N/A ATGTTTGTTTAGTGGA Deoxy, MOE, and cEt 475607 5622 4622 562021 N/A N/A CTAACATTATGTTTGT Deoxy, MOE, and cEt 05615 5630 4623 562022 N/A N/A GCACTACTAACATTAT Deoxy, MOE, and cEt 425621 5636 4624 562023 N/A N/A TTAGCAGCACTACTAA Deoxy, MOE, and cEt 355627 5642 4625 562024 N/A N/A AACCTTTTAGCAGCAC Deoxy, MOE, and cEt 765633 5648 189 562025 N/A N/A TTGATAAAAAACCTTT Deoxy, MOE, and cEt 0 56425657 4626 562026 N/A N/A CAAAAGTAGTTGATAA Deoxy, MOE, and cEt 0 56515666 4627 562027 N/A N/A GGAAACCAAAAGTAGT Deoxy, MOE, and cEt 28 56575672 4628 562028 N/A N/A GAAAGTATGGAAACCA Deoxy, MOE, and cEt 52 56655680 4629 562029 N/A N/A ACATCATAAGAAGGAA Deoxy, MOE, and cEt 8 56785693 4630 562030 N/A N/A TCATAGTAAAAGATAT Deoxy, MOE, and cEt 0 57185733 4631 562031 N/A N/A TCATTTAATCATAGTA Deoxy, MOE, and cEt 7 57265741 4632 562032 N/A N/A GCAGGTTCATTTAATC Deoxy, MOE, and cEt 56 57325747 4633 562033 N/A N/A GTAACATTTTGCTTTG Deoxy, MOE, and cEt 44 57525767 4634 562034 N/A N/A ATATTACTATAGTAAC Deoxy, MOE, and cEt 4 57635778 4635 562035 N/A N/A CAATGTATATTACTAT Deoxy, MOE, and cEt 19 57695784 4636 562036 N/A N/A TAGACACAATGTATAT Deoxy, MOE, and cEt 17 57755790 4637 562037 N/A N/A GGTTTCTTCACACATT Deoxy, MOE, and cEt 63 57995814 4638 562038 N/A N/A CTCAGAAATTCATTGT Deoxy, MOE, and cEt 36 58185833 4639 562039 N/A N/A CTTCTTCCAACTCAGA Deoxy, MOE, and cEt 56 58285843 4640 562040 N/A N/A CTAACTCTTCTTCCAA Deoxy, MOE, and cEt 39 58345849 4641 562041 N/A N/A AATGATCTAACTCTTC Deoxy, MOE, and cEt 33 58405855 4642 562042 N/A N/A GAAAGTTAAATGATCT Deoxy, MOE, and cEt 3 58485863 4643 562043 N/A N/A ATCTTAAAGTTACTTA Deoxy, MOE, and cEt 56 59005915 4644 562044 N/A N/A TATGTGATCTTAAAGT Deoxy, MOE, and cEt 5 59065921 4645 562045 N/A N/A AGTAACTATGTGATCT Deoxy, MOE, and cEt 60 59125927 4646 562046 N/A N/A CTACTAAGTAACTATG Deoxy, MOE, and cEt 0 59185933 4647 562047 N/A N/A TCTTTTCTACTAAGTA Deoxy, MOE, and cEt 18 59245939 4648 562048 N/A N/A TATTACTCTTTTCTAC Deoxy, MOE, and cEt 3 59305945 4649 562049 N/A N/A GCTGGGTATTACTCTT Deoxy, MOE, and cEt 76 59365951 4650 562050 N/A N/A TTGCTTGCTGGGTATT Deoxy, MOE, and cEt 77 59425957 190 562051 N/A N/A TAAAGTTTGCTTGCTG Deoxy, MOE, and cEt 58 59485963 4651 562052 N/A N/A CTATTGTAAAGTTTGC Deoxy, MOE, and cEt 16 59545969 4652 562053 N/A N/A AAGGATCTATTGTAAA Deoxy, MOE, and cEt 5 59605975 4653 562054 N/A N/A CTTATTTAAAAGGATC Deoxy, MOE, and cEt 0 59695984 4654 562055 N/A N/A TAGGACCTTATTTAAA Deoxy, MOE, and cEt 0 59755990 4655 562056 N/A N/A ATTTCCTAGGACCTTA Deoxy, MOE, and cEt 10 59815996 4656 562057 N/A N/A CATGAATGATATTTCC Deoxy, MOE, and cEt 39 59916006 4657 562058 N/A N/A TGCTGGCATGAATGAT Deoxy, MOE, and cEt 62 59976012 4658 562059 N/A N/A TTTTGATGCTGGCATG Deoxy, MOE, and cEt 74 60036018 4659 562060 N/A N/A TTAGTTTTTTGATGCT Deoxy, MOE, and cEt 25 60096024 4660 562061 N/A N/A GCATTATTAGTGTTAG Deoxy, MOE, and cEt 44 60216036 4661 562062 N/A N/A TATCTTGCATTATTAG Deoxy, MOE, and cEt 35 60276042 4662 562063 N/A N/A ATATAATATCTTGCAT Deoxy, MOE, and cEt 0 60336048 4663 562064 N/A N/A CATTGACAGTAAGAAA Deoxy, MOE, and cEt 0 60576072 4664 562065 N/A N/A AGTTTTTCTCATTGAC Deoxy, MOE, and cEt 62 60666081 4665 562143 N/A N/A ATGGATATCTCTTAAC Deoxy, MOE, and cEt 18 68696884 4666 562144 N/A N/A TATTTGATGGATATCT Deoxy, MOE, and cEt 35 68756890 4667 562145 N/A N/A ACATTGTATTTGATGG Deoxy, MOE, and cEt 41 68816896 4668 562146 N/A N/A GTTGATACATTGTATT Deoxy, MOE, and cEt 8 68876902 4669 562147 N/A N/A GTTTAGGTTGATACAT Deoxy, MOE, and cEt 35 68936908 4670 562148 N/A N/A CATCCAGTTTAGGTTG Deoxy, MOE, and cEt 59 68996914 4671 562149 N/A N/A CCCCAGCATCCAGTTT Deoxy, MOE, and cEt 37 69056920 4672 562150 N/A N/A AAAGAACCCCAGCATC Deoxy, MOE, and cEt 35 69116926 4673 562151 N/A N/A GTGTAAAAAGAACCCC Deoxy, MOE, and cEt 33 69176932 4674 562152 N/A N/A TATAGGGTGTAAAAAG Deoxy, MOE, and cEt 0 69236938 4675 562153 N/A N/A GTCTTTTATAGGGTGT Deoxy, MOE, and cEt 75 69296944 191 562154 N/A N/A AGGTATGTCTTTTATA Deoxy, MOE, and cEt 21 69356950 4676 562155 N/A N/A TTGTCTTAGGTATGTC Deoxy, MOE, and cEt 84 69426957 192 562156 N/A N/A CTCTGATTGTCTTAGG Deoxy, MOE, and cEt 77 69486963 193 562157 N/A N/A GTATTTCTCTGATTGT Deoxy, MOE, and cEt 77 69546969 194 562158 N/A N/A AGTCCATATTTGTATT Deoxy, MOE, and cEt 49 69656980 4677 562159 N/A N/A TAATCAAGTCCATATT Deoxy, MOE, and cEt 19 69716986 4678 562160 N/A N/A ATCTAATAATCAAGTC Deoxy, MOE, and cEt 5 69776992 4679 562161 N/A N/A CCTTCTATATTATCTA Deoxy, MOE, and cEt 38 69887003 4680 562162 N/A N/A TAATAAACCTTCTATA Deoxy, MOE, and cEt 8 69957010 4681 562163 N/A N/A GATCACATCTAAGAAA Deoxy, MOE, and cEt 25 70137028 4682 562164 N/A N/A TACCATGATCACATCT Deoxy, MOE, and cEt 66 70197034 4683 562165 N/A N/A CTGCAATACCATGATC Deoxy, MOE, and cEt 54 70257040 4684 562166 N/A N/A GTTCTCCTTTAAAACT Deoxy, MOE, and cEt 0 70397054 4685 562167 N/A N/A GAGATTGTTCTCCTTT Deoxy, MOE, and cEt 7 70457060 4686 562168 N/A N/A AAACAGGAGATTGTTC Deoxy, MOE, and cEt 6 70517066 4687 562169 N/A N/A TCTCTTAAACAGGAGA Deoxy, MOE, and cEt 1 70577072 4688 562170 N/A N/A CATGTATCTCTTAAAC Deoxy, MOE, and cEt 40 70637078 4689 562171 N/A N/A CGTAAATATTTCAGCA Deoxy, MOE, and cEt 30 70777092 4690 562172 N/A N/A TAACTCCGTAAATATT Deoxy, MOE, and cEt 0 70837098 4691 562173 N/A N/A GACCTTTAACTCCGTA Deoxy, MOE, and cEt 68 70897104 4692 562174 N/A N/A TCCAGTGACCTTTAAC Deoxy, MOE, and cEt 6 70957110 4693 562175 N/A N/A CACCAGTCTGGAGTCC Deoxy, MOE, and cEt 52 71087123 4694 562176 N/A N/A TTCTATCACCAGTCTG Deoxy, MOE, and cEt 67 71147129 4695 562177 N/A N/A ATCTTACCAAACTATT Deoxy, MOE, and cEt 23 71717186 4696 562178 N/A N/A AGAATCATCTTACCAA Deoxy, MOE, and cEt 55 71777192 4697 562179 N/A N/A GAATGTAAGAATCATC Deoxy, MOE, and cEt 0 71847199 4698 562180 N/A N/A GTGTTATTTAAGAATG Deoxy, MOE, and cEt 0 71957210 4699 562181 N/A N/A TTTTTCTTAGATGGCG Deoxy, MOE, and cEt 82 72107225 195 562182 N/A N/A GTTTATGTTAAAGCAT Deoxy, MOE, and cEt 8 7225 72404700 562183 N/A N/A AGTAATGTTTATGTTA Deoxy, MOE, and cEt 4 7231 72464701 562184 N/A N/A GTAGCATTTTTTCAGT Deoxy, MOE, and cEt 58 7244 72594702 562185 N/A N/A GCAAATGTAGCATTTT Deoxy, MOE, and cEt 61 7250 72654703 562186 N/A N/A GTTGTGGCAAATGTAG Deoxy, MOE, and cEt 32 7256 72714704 562187 N/A N/A TATGAAGTTGTGGCAA Deoxy, MOE, and cEt 54 7262 72774705 562188 N/A N/A GATTTCACTTGACATT Deoxy, MOE, and cEt 19 7279 72944706 562189 N/A N/A GCTTGAGATTTCACTT Deoxy, MOE, and cEt 42 7285 73004707 562190 N/A N/A TTTGGAGCTTGAGATT Deoxy, MOE, and cEt 22 7291 73064708 562191 N/A N/A AATATCTTTGGAGCTT Deoxy, MOE, and cEt 36 7297 73124709 562192 N/A N/A AGGAATAATATCTTTG Deoxy, MOE, and cEt 5 7303 73184710 562193 N/A N/A ATTTAGTAATAGGAAT Deoxy, MOE, and cEt 5 7313 73284711 562194 N/A N/A CATCAGATTTAGTAAT Deoxy, MOE, and cEt 0 7319 73344712 562195 N/A N/A GTTATTACATCAGATT Deoxy, MOE, and cEt 23 7326 73414713 562196 N/A N/A GCCTAGAATCAATAAA Deoxy, MOE, and cEt 8 7344 73594714 562197 N/A N/A AGGAATGCCTAGAATC Deoxy, MOE, and cEt 2 7350 73654715 562198 N/A N/A TTCAGCAGGAATGCCT Deoxy, MOE, and cEt 46 7356 73714716 562199 N/A N/A TTACCTGATATAACAT Deoxy, MOE, and cEt 41 7460 74754717 562200 N/A N/A CAGGTTTTACCTGATA Deoxy, MOE, and cEt 31 7466 74814718 562201 N/A N/A CTTAGACAGGTTTTAC Deoxy, MOE, and cEt 41 7472 74874719 562202 N/A N/A ATTCTCCTTAGACAGG Deoxy, MOE, and cEt 37 7478 74934720 562203 N/A N/A CTGTCTATTCTCCTTA Deoxy, MOE, and cEt 53 7484 74994721 562204 N/A N/A TAACTACTGTCTATTC Deoxy, MOE, and cEt 5 7490 75054722 562205 N/A N/A TTGAACTAACTACTGT Deoxy, MOE, and cEt 3 7496 75114723 562206 N/A N/A AGTAAGTTGAACTAAC Deoxy, MOE, and cEt 11 7502 75174724 562207 N/A N/A GTAATGAGTAAGTTGA Deoxy, MOE, and cEt 37 7508 75234725 562208 N/A N/A TAATCTTCCTAATACG Deoxy, MOE, and cEt 5 7523 75384726 562209 N/A N/A ACCAGGTTAATCTTCC Deoxy, MOE, and cEt 71 7530 75454727 562210 N/A N/A ATGATAACCAGGTTAA Deoxy, MOE, and cEt 42 7536 75514728 562211 N/A N/A CGAATACTCATATATA Deoxy, MOE, and cEt 20 7576 75914729 562212 N/A N/A TTTATACGAATACTCA Deoxy, MOE, and cEt 17 7582 75974730 562213 N/A N/A ATTATATTTATACGAA Deoxy, MOE, and cEt 0 7588 76034731 562214 N/A N/A GGTAAAAGTATTATAT Deoxy, MOE, and cEt 0 7597 76124732 562215 N/A N/A GAGAATATTGAGTAAA Deoxy, MOE, and cEt 9 7624 76394733 562216 N/A N/A CAGATTATTTTAGAGG Deoxy, MOE, and cEt 16 7645 76604734 562217 N/A N/A TCACTTCAGATTATTT Deoxy, MOE, and cEt 34 7651 76664735 562218 N/A N/A TAATAGTCACTTCAGA Deoxy, MOE, and cEt 33 7657 76724736 562219 N/A N/A TATTGATAATAGTCAC Deoxy, MOE, and cEt 1 7663 76784737 562297 N/A N/A TACTATTTGTAATCAA Deoxy, MOE, and cEt 0 8493 85084738 562298 N/A N/A CTTGCTTATTTTACTA Deoxy, MOE, and cEt 24 8504 85194739 562299 N/A N/A CATCTGTTATTTTATC Deoxy, MOE, and cEt 0 8519 85344740 562300 N/A N/A ATGTGCTTTTTGGATT Deoxy, MOE, and cEt 20 8540 85554741 562301 N/A N/A GGATTTTTGTATGTGC Deoxy, MOE, and cEt 64 8550 85654742 562302 N/A N/A CATCATTCATGGATTT Deoxy, MOE, and cEt 55 8560 85754743 562303 N/A N/A CTTAGACATCATTCAT Deoxy, MOE, and cEt 32 8566 85814744 562304 N/A N/A TGAGTACTTAGACATC Deoxy, MOE, and cEt 58 8572 85874745 562305 N/A N/A TATAAGTGAGTACTTA Deoxy, MOE, and cEt 3 8578 85934746 562306 N/A N/A CTACTTTATAAGTGAG Deoxy, MOE, and cEt 0 8584 85994747 562307 N/A N/A TGAATGTCTTCTACTT Deoxy, MOE, and cEt 42 8594 86094748 562308 N/A N/A TATAATAATGAATGTC Deoxy, MOE, and cEt 2 8602 86174749 562309 N/A N/A GTACTGAGCATTTAAA Deoxy, MOE, and cEt 24 8625 86404750 562310 N/A N/A CAAATAGTACTGAGCA Deoxy, MOE, and cEt 48 8631 86464751 562311 N/A N/A AATGGTCAAATAGTAC Deoxy, MOE, and cEt 0 8637 86524752 562312 N/A N/A GTAGTTTGAATACAAA Deoxy, MOE, and cEt 9 8660 86754753 562313 N/A N/A TCACTGGTAGTTTGAA Deoxy, MOE, and cEt 56 8666 86814754 562314 N/A N/A GGGCTTTCACTGGTAG Deoxy, MOE, and cEt 70 8672 8687196 562315 N/A N/A TAGGTAGGGCTTTCAC Deoxy, MOE, and cEt 50 8678 86934755 562316 N/A N/A ACCTTCTAGGTAGGGC Deoxy, MOE, and cEt 47 8684 86994756 562317 N/A N/A GAGTATACCTTCTAGG Deoxy, MOE, and cEt 38 8690 87054757 562318 N/A N/A ATCACTGAGTATACCT Deoxy, MOE, and cEt 61 8696 87114758 562319 N/A N/A AAACTTATCACTGAGT Deoxy, MOE, and cEt 0 8702 87174759 562320 N/A N/A GCTACAAAACTTATCA Deoxy, MOE, and cEt 8 8708 87234760 562321 N/A N/A TTTGGAGCTACAAAAC Deoxy, MOE, and cEt 0 8714 87294761 562322 N/A N/A AGAAGATTTGGAGCTA Deoxy, MOE, and cEt 24 8720 87354762 562323 N/A N/A ACTATTAGAAGATTTG Deoxy, MOE, and cEt 0 8726 87414763 562324 N/A N/A ACACTCACTATTAGAA Deoxy, MOE, and cEt 0 8732 87474764 562325 N/A N/A AGCCTTTTATTTTGGG Deoxy, MOE, and cEt 37 8751 87664765 562326 N/A N/A CCTGTCAGCCTTTTAT Deoxy, MOE, and cEt 0 8757 87724766 562327 N/A N/A GACTTACCTGTCAGCC Deoxy, MOE, and cEt 47 8763 87784767 562328 N/A N/A ATTCTCGACTTACCTG Deoxy, MOE, and cEt 12 8769 87844768 562329 N/A N/A GTGAGTATTCTCGACT Deoxy, MOE, and cEt 25 8775 87904769 562330 N/A N/A AATTAAGTGAGTATTC Deoxy, MOE, and cEt 0 8781 87964770 562331 N/A N/A TACCAGAATTAAGTGA Deoxy, MOE, and cEt 0 8787 88024771 562332 N/A N/A GCTTTCTTACCAGAAT Deoxy, MOE, and cEt 23 8794 88094772 562333 N/A N/A TGGGTTGCTTTCTTAC Deoxy, MOE, and cEt 0 8800 88154773 562334 N/A N/A TACAAGTACAAATGGG Deoxy, MOE, and cEt 36 8812 88274774 562335 N/A N/A GGTAAATACAAGTACA Deoxy, MOE, and cEt 19 8818 88334775 562336 N/A N/A ATTGCTGGTAAATACA Deoxy, MOE, and cEt 13 8824 88394776 562337 N/A N/A TTAAGGATTGCTGGTA Deoxy, MOE, and cEt 43 8830 88454777 562338 N/A N/A GCTTCATTTTAAGGAT Deoxy, MOE, and cEt 12 8838 88534778 562339 N/A N/A GTAGGAAGCTTCATTT Deoxy, MOE, and cEt 23 8845 88604779 562340 N/A N/A GAGTTAGTAGGAAGCT Deoxy, MOE, and cEt 58 8851 88664780 562341 N/A N/A GCTATTGAGTTAGTAG Deoxy, MOE, and cEt 21 8857 88724781 562342 N/A N/A CTTATTGCTATTGAGT Deoxy, MOE, and cEt 34 8863 88784782 562343 N/A N/A TATTGTCTTATTGCTA Deoxy, MOE, and cEt 17 8869 88844783 562344 N/A N/A ATTCACTATTGTCTTA Deoxy, MOE, and cEt 22 8875 88904784 562345 N/A N/A ATCACAATCCTTTTTA Deoxy, MOE, and cEt 18 8925 89404785 562346 N/A N/A TTCTTCATCACAATCC Deoxy, MOE, and cEt 43 8931 89464786 562347 N/A N/A AGATTGTTCTTCATCA Deoxy, MOE, and cEt 35 8937 89524787 562348 N/A N/A TATAAATAGATTGTTC Deoxy, MOE, and cEt 10 8944 89594788 562349 N/A N/A GGTTCTTAATAACTTT Deoxy, MOE, and cEt 31 9011 90264789 562350 N/A N/A AAGCATGGTTCTTAAT Deoxy, MOE, and cEt 12 9017 90324790 562351 N/A N/A CTTTGTAGAAAAAGAC Deoxy, MOE, and cEt 0 9066 90814791 562352 N/A N/A TATGCTTTCTTTGTAG Deoxy, MOE, and cEt 26 9074 90894792 562353 N/A N/A CTTAATGTATGCTTTC Deoxy, MOE, and cEt 55 9081 90964793 562354 N/A N/A GTATTTGCTTAATGTA Deoxy, MOE, and cEt 0 9088 91034794 562355 N/A N/A CCTTTGGTATTTGCTT Deoxy, MOE, and cEt 54 9094 91094795 562356 N/A N/A ACCTGGCCTTTGGTAT Deoxy, MOE, and cEt 0 9100 91154796 562357 N/A N/A ATGTAAACCTGGCCTT Deoxy, MOE, and cEt 1 9106 91214797 562358 N/A N/A CTTCAAATGTAAACCT Deoxy, MOE, and cEt 0 9112 91274798 562359 N/A N/A GTAATAATAATGTCAC Deoxy, MOE, and cEt 0 9131 91464799 562360 N/A N/A AGACTTGAGTAATAAT Deoxy, MOE, and cEt 0 9139 91544800 562361 N/A N/A TCCTAGAGACTTGAGT Deoxy, MOE, and cEt 25 9145 91604801 562362 N/A N/A AAGTATTCCTAGAGAC Deoxy, MOE, and cEt 28 9151 91664802 562363 N/A N/A TGTGTTAAGTATTCCT Deoxy, MOE, and cEt 50 9157 91724803 562364 N/A N/A AAGAGATGTGTTAAGT Deoxy, MOE, and cEt 21 9163 91784804 562365 N/A N/A ACAGTCAAGAGATGTG Deoxy, MOE, and cEt 74 9169 9184197 562366 N/A N/A CCATATACAGTCAAGA Deoxy, MOE, and cEt 49 9175 91904805 562367 N/A N/A TAACATCCATATACAG Deoxy, MOE, and cEt 16 9181 91964806 562368 N/A N/A CTATTTATTAACATCC Deoxy, MOE, and cEt 2 9189 92044807 562369 N/A N/A TGTCAGCTATTTATTA Deoxy, MOE, and cEt 22 9195 92104808 562370 N/A N/A CTTTACTGTCAGCTAT Deoxy, MOE, and cEt 56 9201 92164809 562371 N/A N/A GATAAACTTTACTGTC Deoxy, MOE, and cEt 37 9207 92224810 562372 N/A N/A CTTTATATGGATAAAC Deoxy, MOE, and cEt 31 9216 92314811 562373 N/A N/A GCAAGTCTTTATATGG Deoxy, MOE, and cEt 62 9222 92374812 560990 709 724 TTCTTGGTGCTCTTGG Deoxy, MOE, and cEt 74 6722 6737111 337487 804 823 CACTTGTATGTTCACCTCTG 5-10-5 MOE 30 7389 7408 28233717 889 908 TGAATTAATGTCCATGGACT 5-10-5 MOE 38 7876 7895 14

Example 121: Antisense Inhibition of Human ANGPTL3 in Hep3B Cells by MOEGapmers

Additional antisense oligonucleotides were designed targeting an ANGPTL3nucleic acid and were tested for their effects on ANGPTL3 mRNA in vitro.Cultured Hep3B cells at a density of 20,000 cells per well weretransfected using electroporation with 4,500 nM antisenseoligonucleotide. After a treatment period of approximately 24 hours, RNAwas isolated from the cells and ANGPTL3 mRNA levels were measured byquantitative real-time PCR. Human primer probe set RTS3492_MGB was usedto measure mRNA levels. ANGPTL3 mRNA levels were adjusted according tototal RNA content, as measured by RIBOGREEN®. Results are presented aspercent inhibition of ANGPTL3, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in the Tablesbelow were designed as 5-10-5 MOE or 3-10-4 MOE gapmers. The 5-10-5 MOEgapmers are 20 nucleosides in length, wherein the central gap segmentcomprises often 2′-deoxynucleosides and is flanked by wing segments onthe 5′ direction and the 3′ direction comprising five nucleosides each.The 3-10-4 MOE gapmers are 17 nucleosides in length, wherein the centralgap segment comprises often 2′-deoxynucleosides and is flanked by wingsegments on the 5′ direction and the 3′ direction comprising three andfour nucleosides respectively. Each nucleoside in the 5′ wing segmentand each nucleoside in the 3′ wing segment has a 2′-MOE modification.Each nucleoside in the 5′ wing segment and each nucleoside in the 3′wing segment has a 2′-MOE modification. The internucleoside linkagesthroughout each gapmer are phosphorothioate (P═S) linkages. All cytosineresidues throughout each gapmer are 5-methylcytosines. “Start site”indicates the 5′-most nucleoside to which the gapmer is targeted in thehuman gene sequence. “Stop site” indicates the 3′-most nucleoside towhich the gapmer is targeted human gene sequence. Each gapmer listed inthe Tables below is targeted to either the human ANGPTL3 mRNA,designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_014495.2) orthe human ANGPTL3 genomic sequence, designated herein as SEQ ID NO: 2(GENBANK Accession No. NT_032977.9 truncated from nucleotides 33032001to 33046000). ‘n/a’ indicates that the antisense oligonucleotide doesnot target that particular gene sequence with 100% complementarity.

TABLE 155 Inhibition of ANGPTL3 mRNA by MOE gapmers targeting SEQ ID NO:1 and 2 SEQ SEQ SEQ SEQ ID NO: ID NO: ID NO: ID NO: ISIS 1 Start 1 Stop% 2 Start 2 Stop SEQ NO Site Site Sequence Motif inhibition Site Site IDNO 582715 N/A N/A CTGGGTATTACTCTTTTCTA 5-10-5 60 5931 5950 4813 582716N/A N/A CTTGCTGGGTATTACTCTTT 5-10-5 59 5935 5954 4814 582717 N/A N/ATGCTTGCTGGGTATTACTCT 5-10-5 59 5937 5956 4815 582718 N/A N/ACATGAATGATATTTCCTAGG 5-10-5 39 5987 6006 4816 582719 N/A N/AGGCATGAATGATATTTCCTA 5-10-5 60 5989 6008 4817 582720 N/A N/ACTGGCATGAATGATATTTCC 5-10-5 46 5991 6010 4818 582721 N/A N/ATGCTGGCATGAATGATATTT 5-10-5 32 5993 6012 4819 582722 N/A N/AAAGTCCATATTTGTATTTCT 5-10-5 50 6962 6981 4820 582723 N/A N/AGCAAATGTAGCATTTTTTCA 5-10-5 32 7246 7265 4821 582724 N/A N/AGGCAAATGTAGCATTTTTTC 5-10-5 55 7247 7266 4822 582725 N/A N/AGTGGCAAATGTAGCATTTTT 5-10-5 62 7249 7268 203 582726 N/A N/ACTGGTCCTTTTAACTTCCAA 5-10-5 40 8366 8385 4823 582727 N/A N/ACCTGGTCCTTTTAACTTCCA 5-10-5 58 8367 8386 4824 582728 N/A N/ATTCCTGGTCCTTTTAACTTC 5-10-5 32 8369 8388 4825 582729 N/A N/ATGCTTAATGTATGCTTTCTT 5-10-5 51 9079 9098 4826 582730 N/A N/ACCGTAAGTTTATCTTCCTTT 5-10-5 58 10136 10155 4827 582731 N/A N/ACCCCGTAAGTTTATCTTCCT 5-10-5 51 10138 10157 4828 582732 N/A N/ACACAAATATGTTCATTCTTA 5-10-5 22 11189 11208 4829 582733 N/A N/AGCCACAAATATGTTCATTCT 5-10-5 71 11191 11210 204 582734 N/A N/AAAACTTTAACTCGATGCCAC 5-10-5 51 11206 11225 4830 582735 N/A N/AATAAACTTTAACTCGATGCC 5-10-5 57 11208 11227 4831 582736 N/A N/AATGCTTGTCAGGCTGTTTAA 5-10-5 56 11311 11330 4832 582737 N/A N/AGTCACCATATAACTTGGGCA 5-10-5 48 11562 11581 4833 582738 N/A N/AAGGTCACCATATAACTTGGG 5-10-5 44 11564 11583 4834 582766 N/A N/AGCTGGGTATTACTCTTT 3-10-4 55 5935 5951 4835 582767 N/A N/AGCATGAATGATATTTCC 3-10-4 4 5991 6007 4836 582768 N/A N/AGGCAAATGTAGCATTTT 3-10-4 33 7250 7266 4837 582769 N/A N/ACTGGTCCTTTTAACTTC 3-10-4 29 8369 8385 4838 582770 N/A N/AGTAAGTTTATCTTCCTT 3-10-4 26 10137 10153 4839 582771 N/A N/AACTTTAACTCGATGCCA 3-10-4 42 11207 11223 4840 582772 N/A N/AAACTTTAACTCGATGCC 3-10-4 55 11208 11224 4841 582773 N/A N/AAAACTTTAACTCGATGC 3-10-4 1 11209 11225 4842 582774 N/A N/AGCTTGTCAGGCTGTTTA 3-10-4 65 11312 11328 208 582775 N/A N/ACACCATATAACTTGGGC 3-10-4 38 11563 11579 4843 582776 N/A N/ATCACCATATAACTTGGG 3-10-4 37 11564 11580 4844 582777 N/A N/AGTCACCATATAACTTGG 3-10-4 31 11565 11581 4845 582702 139 158CTTGATTTTGGCTCTGGAGA 5-10-5 53 3243 3262 4846 582739 140 156TGATTTTGGCTCTGGAG 3-10-4 41 3244 3260 4847 582703 141 160ATCTTGATTTTGGCTCTGGA 5-10-5 64 3245 3264 198 582740 305 321ACTGGTTTGCAGCGATA 3-10-4 58 3409 3425 4848 582704 306 325TTTCACTGGTTTGCAGCGAT 5-10-5 60 3410 3429 4849 582741 306 322CACTGGTTTGCAGCGAT 3-10-4 57 3410 3426 4850 582742 307 323TCACTGGTTTGCAGCGA 3-10-4 60 3411 3427 4851 582705 706 725GTTCTTGGTGCTCTTGGCTT 5-10-5 78 6719 6738 199 544120 707 726AGTTCTTGGTGCTCTTGGCT 5-10-5 75 6720 6739 15 582743 707 723TCTTGGTGCTCTTGGCT 3-10-4 63 6720 6736 205 582706 708 727TAGTTCTTGGTGCTCTTGGC 5-10-5 69 6721 6740 200 582744 708 724TTCTTGGTGCTCTTGGC 3-10-4 51 6721 6737 4852 582745 709 725GTTCTTGGTGCTCTTGG 3-10-4 50 6722 6738 4853 337487 804 823CACTTGTATGTTCACCTCTG 5-10-5 25 7389 7408 28 233717 889 908TGAATTAATGTCCATGGACT 5-10-5 22 7876 7895 14 582707 1054 1073TTGTCTTTCCAGTCTTCCAA 5-10-5 42 9629 9648 4854 582708 1056 1075TGTTGTCTTTCCAGTCTTCC 5-10-5 52 9631 9650 4855 582746 1140 1156CATTGCCAGTAATCGCA 3-10-4 53 9715 9731 4856 582747 1141 1157ACATTGCCAGTAATCGC 3-10-4 61 9716 9732 4857 582748 1142 1158GACATTGCCAGTAATCG 3-10-4 34 9717 9733 4858 582709 1194 1213CTTTGTGATCCCAAGTAGAA 5-10-5 28 9769 9788 4859 582749 1195 1211TTGTGATCCCAAGTAGA 3-10-4 16 9770 9786 4860 582710 1196 1215TGCTTTGTGATCCCAAGTAG 5-10-5 54 9771 9790 4861 582750 1196 1212TTTGTGATCCCAAGTAG 3-10-4 19 9771 9787 4862 582751 1197 1213CTTTGTGATCCCAAGTA 3-10-4 32 9772 9788 4863 582752 1260 1276CACACTCATCATGCCAC 3-10-4 42 10232 10248 4864 582711 1268 1287GTTGTTTTCTCCACACTCAT 5-10-5 51 10240 10259 4865 582712 1270 1289AGGTTGTTTTCTCCACACTC 5-10-5 63 10242 10261 201 582753 1307 1323AGATTTTGCTCTTGGTT 3-10-4 54 10279 10295 4866 582754 1308 1324TAGATTTTGCTCTTGGT 3-10-4 52 10280 10296 4867 582755 1309 1325TTAGATTTTGCTCTTGG 3-10-4 44 10281 10297 4868 582756 1310 1326CTTAGATTTTGCTCTTG 3-10-4 34 10282 10298 4869 567320 1487 1506CCAGATTATTAGACCACATT 5-10-5 77 10459 10478 93 582757 1488 1504AGATTATTAGACCACAT 3-10-4 0 10460 10476 4870 582758 1489 1505CAGATTATTAGACCACA 3-10-4 39 10461 10477 4871 582759 1490 1506CCAGATTATTAGACCAC 3-10-4 63 10462 10478 206 582760 1491 1507ACCAGATTATTAGACCA 3-10-4 31 10463 10479 4872 582761 1763 1779GCTCATATGATGCCTTT 3-10-4 71 10735 10751 207 582713 1906 1925ACACATACTCTGTGCTGACG 5-10-5 68 10878 10897 202 582762 1907 1923ACATACTCTGTGCTGAC 3-10-4 57 10879 10895 4873 582714 1908 1927TTACACATACTCTGTGCTGA 5-10-5 49 10880 10899 4874 582763 2071 2087CTTAGTAGTCATCTCCA 3-10-4 49 11043 11059 4875 582764 2072 2088ACTTAGTAGTCATCTCC 3-10-4 53 11044 11060 4876 582765 2073 2089GACTTAGTAGTCATCTC 3-10-4 36 11045 11061 4877

Example 122: Dose-Dependent Antisense Inhibition of Human ANGPTL3 inHep3B Cells

Deoxy, MOE, and cEt oligonucleotides from the studies described aboveexhibiting significant in vitro inhibition of ANGPTL3 mRNA were selectedand tested at various doses in Hep3B cells. ISIS 233717 and ISIS 337847,both 5-10-5 MOE gapmers, were also included in the studies. Theantisense oligonucleotides were tested in a series of experiments thathad similar culture conditions. The results of each experiment arepresented in separate tables below.

Cells were plated at a density of 20,000 cells per well and transfectedusing electroporation with 0.813 μM, 1.625 μM, 3.25 μM, 6.500 μM and13.00 μM concentrations of antisense oligonucleotide, as specified inthe Table below. After a treatment period of approximately 16 hours, RNAwas isolated from the cells and ANGPTL3 mRNA levels were measured byquantitative real-time PCR. Human primer probe set RTS3492_MGB was usedto measure mRNA levels. ANGPTL3 mRNA levels were adjusted according tototal RNA content, as measured by RIBOGREEN. Results are presented aspercent inhibition of ANGPTL3, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented. ANGPTL3 mRNA levels were significantly reduced in adose-dependent manner in antisense oligonucleotide treated cells.

TABLE 156 IC₅₀ SEQ ID ISIS No 0.813 μM 1.625 μM 3.25 μM 6.50 μM 13.00 μM(μM) NO 233717 0 27 43 66 79 4.4 14 337487 26 49 63 85 94 2.0 28 55927754 68 70 82 91 <0.8 110 560990 36 61 74 90 96 1.2 111 560992 60 67 76 8193 <0.8 112 561010 71 77 82 86 94 <0.8 113 561011 80 87 91 95 97 <0.8114 561022 75 79 84 89 93 <0.8 115 561025 68 82 81 91 96 <0.8 116 56102672 85 85 89 90 <0.8 117 561208 63 80 87 92 93 <0.8 118 561320 47 60 8692 96 0.8 119 561343 45 59 79 86 93 0.9 120 561345 38 59 80 88 95 1.1121 561347 53 63 84 88 97 <0.8 122

TABLE 157 IC₅₀ SEQ ID ISIS No 0.813 μM 1.625 μM 3.25 μM 6.50 μM 13.00 μM(μM) NO 233717 7 19 55 60 77 4.2 14 337487 33 44 69 83 88 2.0 28 56099036 64 81 87 95 1.1 111 561452 58 69 75 85 88 <0.8 123 561458 69 77 84 9194 <0.8 124 561460 54 50 72 79 85 <0.8 125 561462 49 72 80 90 92 <0.8126 561463 63 79 84 92 93 <0.8 127 561478 56 53 80 86 91 <0.8 128 56148246 69 80 86 91 <0.8 129 561486 56 73 80 91 92 <0.8 130 561487 82 87 8890 93 <0.8 131 561500 52 60 71 80 91 <0.8 132 561504 49 72 85 91 93 <0.8133 561621 68 76 85 91 94 <0.8 134

TABLE 158 IC₅₀ SEQ ID ISIS No 0.813 μM 1.625 μM 3.25 μM 6.50 μM 13.00 μM(μM) NO 233717 28 35 48 56 60 4.7 14 337487 43 58 72 82 89 1.0 28 56099057 73 82 86 96 <0.8 111 561620 51 74 80 85 88 <0.8 135 561622 63 73 8588 87 <0.8 136 561628 48 69 77 79 80 <0.8 137 561631 60 75 84 86 90 <0.8138 561644 59 69 77 85 83 <0.8 139 561646 67 81 84 91 92 <0.8 140 56164970 76 85 89 89 <0.8 141 561650 78 85 88 90 91 <0.8 142 561770 66 81 7988 91 <0.8 143 561781 65 67 80 81 91 <0.8 144 561791 68 73 83 82 85 <0.8145 561918 63 71 81 86 92 <0.8 146

TABLE 159 IC₅₀ SEQ ID ISIS No 0.813 μM 1.625 μM 3.25 μM 6.50 μM 13.00 μM(μM) NO 233717 21 26 47 62 69 4.2 14 337487 35 54 73 82 92 1.0 28 56099042 76 81 88 96 <0.8 111 562078 55 85 86 91 93 <0.8 147 562086 64 83 8792 93 <0.8 148 562103 72 83 90 90 94 <0.8 149 562110 66 80 83 89 92 <0.8150 562375 56 61 63 84 90 <0.8 151 562387 67 75 81 90 88 <0.8 152 56239660 71 80 80 85 <0.8 153 562415 66 73 77 77 81 <0.8 154 562433 68 84 8690 91 <0.8 155 562436 78 87 87 91 94 <0.8 156 562439 55 66 78 82 93 <0.8157 562442 55 57 60 76 86 <0.8 158

Example 123: Dose-Dependent Antisense Inhibition of Human ANGPTL3 inHep3B Cells

Deoxy, MOE, and cEt oligonucleotides from the studies described aboveexhibiting significant in vitro inhibition of ANGPTL3 mRNA were selectedand tested at various doses in Hep3B cells. ISIS 337847, a 5-10-5 MOEgapmer, was also included in the studies. The antisense oligonucleotideswere tested in a series of experiments that had similar cultureconditions. The results of each experiment are presented in separatetables below.

Cells were plated at a density of 20,000 cells per well and transfectedusing electroporation with 0.160 μM, 0.481 μM, 1.444 μM, 4.333 μM and13.00 μM concentrations of antisense oligonucleotide, as specified inthe Table below. After a treatment period of approximately 16 hours, RNAwas isolated from the cells and ANGPTL3 mRNA levels were measured byquantitative real-time PCR. Human primer probe set RTS3492_MGB was usedto measure mRNA levels. ANGPTL3 mRNA levels were adjusted according tototal RNA content, as measured by RIBOGREEN®. Results are presented aspercent inhibition of ANGPTL3, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented. ANGPTL3 mRNA levels were significantly reduced in adose-dependent manner in antisense oligonucleotide treated cells.

TABLE 160 IC₅₀ SEQ ID ISIS No 0.160 μM 0.481 μM 1.444 μM 4.333 μM 13.00μM (μM) NO 337487 0 18 24 49 73 4.1 28 560990 2 27 39 59 80 2.0 111561076 20 33 59 73 89 1.1 159 561079 24 39 51 72 84 1.0 160 561084 7 1746 66 87 1.9 161 561085 21 35 55 69 86 1.2 162 561123 20 39 52 72 87 1.1163 561241 13 22 41 68 86 2.0 164 561256 12 22 35 54 82 2.6 165 56126022 16 34 54 82 2.6 166 561277 21 21 37 59 69 2.9 167 561288 6 8 23 36 686.9 168 561418 25 36 61 79 86 0.9 169 561436 21 40 61 77 88 0.9 170561443 18 32 52 82 88 1.1 171

TABLE 161 IC₅₀ SEQ ID ISIS No 0.160 μM 0.481 μM 1.444 μM 4.333 μM 13.00μM (μM) NO 337487 0 8 21 52 81 3.7 28 560990 6 14 40 61 74 3.0 111561398 3 9 22 64 79 3.0 172 561400 11 28 50 65 83 1.7 173 561528 2 39 5974 84 1.3 174 561565 18 43 58 75 83 1.0 175 561566 21 29 54 71 79 1.4176 561567 16 35 56 67 78 1.4 177 561571 18 32 60 80 86 1.1 178 56157611 12 42 65 77 2.4 179 561689 16 27 52 76 80 1.4 180 561698 1 24 31 6174 2.9 181 561699 2 19 48 65 81 2.0 182 561722 14 34 59 72 85 1.2 183561723 7 31 69 71 75 1.4 184

TABLE 162 IC₅₀ SEQ ID ISIS No 0.160 μM 0.481 μM 1.444 μM 4.333 μM 13.00μM (μM) NO 337487 14 9 9 47 72 5.9 28 560990 13 26 39 58 81 2.0 111561888 16 19 46 72 84 1.7 185 561897 6 31 50 67 82 2.0 186 561996 19 3149 59 83 1.6 187 562001 22 46 57 67 89 0.9 188 562024 17 29 59 71 83 1.3189 562050 21 38 46 62 74 1.6 190 562153 22 35 42 61 71 2.0 191 56215529 29 50 72 84 1.2 192 562156 15 17 39 60 82 2.3 193 562157 14 15 43 5475 3.0 194 562181 24 34 58 73 80 1.1 195 562314 22 30 42 54 64 3.1 196562365 25 27 46 64 77 1.7 197

Example 124: Dose-Dependent Antisense Inhibition of Human ANGPTL3 inHep3B Cells by MOE Gapmers

MOE gapmers from the Examples above exhibiting significant in vitroinhibition of ANGPTL3 mRNA were selected and tested at various doses inHep3B cells. Cells were plated at a density of 20,000 cells per well andtransfected using electroporation with 0.160 μM, 0.481 μM, 1.444 μM,4.333 μM and 13.00 μM concentrations of antisense oligonucleotide, asspecified in the Table below. After a treatment period of approximately16 hours, RNA was isolated from the cells and ANGPTL3 mRNA levels weremeasured by quantitative real-time PCR. Human primer probe setRTS3492_MGB was used to measure mRNA levels. ANGPTL3 mRNA levels wereadjusted according to total RNA content, as measured by RIBOGREEN.Results are presented as percent inhibition of ANGPTL3, relative tountreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented. ANGPTL3 mRNA levels were significantly reduced in adose-dependent manner in antisense oligonucleotide treated cells.

TABLE 163 IC₅₀ SEQ ID ISIS No Motif 0.16 μM 0.48 μM 1.44 μM 4.33 μM13.00 μM (μM) NO 233717 5-10-5 0 3 12 38 64 8.0 14 337487 5-10-5 0 0 1530 66 8.0 28 544120 5-10-5 10 37 62 81 94 1.0 15 567320 5-10-5 0 30 6784 95 1.1 93 582703 5-10-5 0 18 47 71 83 2.0 198 582705 5-10-5 22 18 4682 93 1.0 199 582706 5-10-5 2 0 32 67 85 2.6 200 582712 5-10-5 0 0 54 7189 2.2 201 582713 5-10-5 25 25 52 75 85 1.2 202 582725 5-10-5 0 3 43 6284 2.7 203 582733 5-10-5 0 30 66 77 87 1.3 204 582743 3-10-4 0 6 37 5187 2.9 205 582759 3-10-4 0 2 51 76 93 2.0 206 582761 3-10-4 4 38 58 7287 1.3 207 582774 3-10-4 5 29 46 72 86 1.6 208

Example 125: Dose-Dependent Antisense Inhibition of Human ANGPTL3 inHep3B Cells by Deoxy, MOE and cEt Oligonucleotides

Deoxy, MOE, and cEt oligonucleotides from the studies described aboveexhibiting significant in vitro inhibition of ANGPTL3 mRNA were selectedand tested at various doses in Hep3B cells. Cells were plated at adensity of 20,000 cells per well and transfected using electroporationwith 0.111 μM, 0.333 μM, 1.00 μM, 3.00 μM and 9.00 μM concentrations ofantisense oligonucleotide, as specified in the Table below. After atreatment period of approximately 16 hours, RNA was isolated from thecells and ANGPTL3 mRNA levels were measured by quantitative real-timePCR. Human primer probe set RTS3492_MGB was used to measure mRNA levels.ANGPTL3 mRNA levels were adjusted according to total RNA content, asmeasured by RIBOGREEN. Results are presented as percent inhibition ofANGPTL3, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented. ANGPTL3 mRNA levels were significantly reduced in adose-dependent manner in antisense oligonucleotide treated cells.

TABLE 164 IC₅₀ SEQ ID ISIS No 0.111 μM 0.333 μM 1.00 μM 3.00 μM 9.00 μM(μM) NO 561011 20 39 65 81 94 0.5 114 561026 23 43 65 84 94 0.5 117561463 26 25 59 76 91 0.7 127 561487 42 61 81 89 95 0.1 131 586661 24 3646 76 92 0.7 209 586669 31 50 68 85 95 0.3 210 586676 26 50 73 83 95 0.3211 586688 4 24 51 82 91 0.9 212 586690 19 39 64 84 95 0.5 213 586691 637 60 81 93 0.7 214 586701 10 32 55 76 90 0.8 215 586702 16 25 55 69 860.9 216 586705 10 30 54 80 89 0.8 217 586707 33 42 71 83 89 0.3 218586718 38 54 72 78 85 0.2 219

TABLE 165 0.111 0.333 1.00 3.00 9.00 IC₅₀ SEQ ISIS No μM μM μM μM μM(μM) ID NO 561011 13 29 41 76 89 1.0 114 561567 20 46 57 75 78 0.7 177586692 32 30 71 85 95 0.4 220 586700 3 46 70 82 95 1.0 221 586708 36 4662 77 86 0.4 222 586744 0 19 54 81 92 1.0 223 586745 35 22 66 78 92 0.5224 586746 14 30 59 82 92 0.7 225 586755 18 22 53 74 90 0.9 226 58676126 26 54 73 90 0.8 227 586787 0 38 64 79 90 0.8 228 586796 12 13 56 8393 0.9 229 586797 4 26 58 82 90 0.9 230 586802 12 28 56 76 81 0.9 231586804 17 40 65 86 93 0.5 232

TABLE 166 0.111 0.333 1.00 3.00 9.00 IC₅₀ SEQ ISIS No μM μM μM μM μM(μM) ID NO 561011 20 48 75 84 94 0.4 114 561026 31 48 70 88 95 0.3 117561463 27 40 67 85 94 0.4 127 561487 41 66 84 91 95 0.1 131 586661 36 4564 82 91 0.3 209 586669 21 55 73 90 96 0.3 210 586676 23 59 77 87 94 0.3211 586688 25 41 70 82 93 0.4 212 586690 16 45 74 86 92 0.5 213 58669113 40 65 86 92 0.6 214 586701 22 49 70 82 93 0.4 215 586702 11 31 58 7692 0.8 216 586705 26 45 66 82 89 0.4 217 586707 28 58 75 85 88 0.3 218586718 33 59 73 80 88 0.2 219

TABLE 167 0.111 0.333 1.00 3.00 9.00 IC₅₀ SEQ ISIS No μM μM μM μM μM(μM) ID NO 561011 23 41 63 82 92 0.5 114 561567 31 44 65 75 83 0.4 177586692 16 58 74 89 93 0.4 220 586700 25 62 75 91 94 0.3 221 586708 36 5372 81 90 0.3 222 586744 30 29 64 75 94 0.6 223 586745 21 44 59 81 89 0.5224 586746 19 48 57 85 87 0.5 225 586755 6 30 59 78 89 0.8 226 586761 1229 59 72 87 0.9 227 586787 27 35 64 84 97 0.5 228 586796 31 40 72 91 950.3 229 586797 36 47 67 82 88 0.3 230 586802 35 32 61 76 90 0.5 231586804 35 50 75 91 91 0.2 232

Example 126: Antisense Inhibition of Human ANGPTL3 in huANGPTL3Transgenic Mice

Antisense oligonucleotides described in the studies above were furtherevaluated for their ability to reduce human ANGPTL3 mRNA transcript inC57Bl/6 mice with the human ANGPTL3 transgene (Tg mice).

Study 1

Female Tg mice were maintained on a 12-hour light/dark cycle. Animalswere acclimated for at least 7 days in the research facility beforeinitiation of the experiment. Antisense oligonucleotides (ASOs) wereprepared in buffered saline (PBS) and sterilized by filtering through a0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS forinjection.

Groups of mice received intraperitoneal injections of 5-10-5 MOE gapmersat a dose of 50 mg/kg once per week for 2 weeks. One group of micereceived subcutaneous injections of PBS once weekly for 2 weeks. ThePBS-injected group served as the control group to which thecorresponding oligonucleotide-treated groups were compared.

RNA Analysis

At the end of the treatment period, RNA was extracted from liver forreal-time PCR analysis of measurement of mRNA expression of ANGPTL3 withRTS3492_MGB. mRNA levels were also measured with human primer probe setRTS 1984 (forward sequence CTTCAATGAAACGTGGGAGAACT, designated herein asSEQ ID NO: 7; reverse sequence TCTCTAGGCCCAACCAAAATTC, designated hereinas SEQ ID NO: 8; probe sequence AAATATGGTTTTGGGAGGCTTGAT, designatedherein as SEQ ID NO: 9). Results are presented as percent change ofmRNA, relative to PBS control, normalized with RIBOGREEN®. As shown inthe Table below, treatment with ISIS antisense oligonucleotides resultedin significant reduction of ANGPTL3 mRNA in comparison to the PBScontrol.

TABLE 168 Percent inhibition of ANGPTL3 mRNA in transgenic mouse liverrelative to the PBS control SEQ ISIS No RTS3492_MGB RTS1984 ID NO 23371091 94 233 233717 49 58 14 337477 76 82 234 337478 52 65 235 337479 53 76236 337487 80 92 28

Protein Analysis

Human ANGPTL3 protein levels were quantified using a commerciallyavailable ELISA kit (Catalog #DANL30 by R&D Systems, Minneapolis, Minn.)with transgenic plasma samples diluted 1:20,000 using the manufacturerdescribed protocol. The results are presented in the Table below. Theresults indicate that treatment with ISIS oligonucleotides resulted inreduced ANGPTL3 protein levels.

TABLE 169 Percent inhibition of plasma protein levels in the transgenicmouse SEQ ISIS No % ID NO 233710 92 233 233717 47 14 337477 68 234337478 36 235 337479 48 236 337487 78 28

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on day 10, plasma levelsof transaminases (ALT and AST) were measured using an automated clinicalchemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The resultsare presented in the Table below. ISIS oligonucleotides that causedchanges in the levels of any of these liver function markers outside theexpected range for antisense oligonucleotides were excluded in furtherstudies.

TABLE 170 Plasma transaminase levels (IU/L) in transgenic mice on day 10SEQ ALT AST ID NO PBS 27 36 ISIS 233710 19 37 233 ISIS 233717 16 32 14ISIS 337477 22 35 234 ISIS 337478 23 49 235 ISIS 337479 21 29 236 ISIS337487 19 35 28

Study 2

Male Tg mice were maintained on a 12-hour light/dark cycle. Animals wereacclimated for at least 7 days in the research facility beforeinitiation of the experiment. Antisense oligonucleotides (ASOs) wereprepared in buffered saline (PBS) and sterilized by filtering through a0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS forinjection.

Groups of mice received intraperitoneal injections of 5-10-5 MOE gapmersat a dose of 50 mg/kg once per week for 2 weeks. One group of micereceived subcutaneous injections of PBS once weekly for 2 weeks. ThePBS-injected groups served as the control groups to which thecorresponding oligonucleotide-treated groups were compared.

RNA Analysis

At the end of the treatment period, RNA was extracted from liver forreal-time PCR analysis of measurement of mRNA expression of ANGPTL3 withRTS 1984. Results are presented as percent change of mRNA, relative toPBS control, normalized with RIBOGREEN. As shown in the Table below,treatment with ISIS antisense oligonucleotides resulted in significantreduction of ANGPTL3 mRNA in comparison to the PBS control.

TABLE 171 Percent inhibition of ANGPTL3 mRNA in transgenic mouse liverrelative to the PBS control SEQ ISIS No % ID NO 233710 81 233 337487 9228 544145 98 16 544162 75 18 544199 97 20 560306 90 34 560400 97 35560401 95 36 560402 98 37 560469 98 38 560735 87 49 567320 95 93 56732193 94

Protein Analysis

Human ANGPTL3 protein levels were quantified using a commerciallyavailable ELISA kit (Catalog #DANL30 by R&D Systems, Minneapolis, Minn.)with transgenic plasma samples diluted 1:20,000 using the manufacturerdescribed protocol. The results are presented in the Table below. Theresults indicate that treatment with ISIS oligonucleotides resulted inreduced ANGPTL3 protein levels.

TABLE 172 Percent inhibition of plasma protein levels in the transgenicmouse SEQ ISIS No % ID NO 233710 96 233 337487 78 28 544145 96 16 54416297 18 544199 98 20 560306 97 34 560400 98 35 560401 97 36 560402 94 37560469 96 38 560735 91 49 567320 98 93 567321 96 94

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on day 8, plasma levelsof transaminases (ALT and AST) were measured using an automated clinicalchemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The resultsare presented in the Table below. ISIS oligonucleotides that causedchanges in the levels of any of these liver function markers outside theexpected range for antisense oligonucleotides were excluded in furtherstudies.

TABLE 173 Plasma transaminase levels (IU/L) in transgenic mice on day 8SEQ ALT AST ID NO PBS 29 44 ISIS 233710 29 47 233 ISIS 337487 22 36 28ISIS 544145 29 45 16 ISIS 544162 31 62 18 ISIS 544199 29 51 20 ISIS560306 23 42 34 ISIS 560400 24 52 35 ISIS 560401 20 38 36 ISIS 560402 2949 37 ISIS 560469 22 50 38 ISIS 560735 20 38 49 ISIS 567320 49 71 93ISIS 567321 20 44 94

Study 3

Male and female Tg mice were maintained on a 12-hour light/dark cycle.Animals were acclimated for at least 7 days in the research facilitybefore initiation of the experiment. Antisense oligonucleotides (ASOs)were prepared in buffered saline (PBS) and sterilized by filteringthrough a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBSfor injection.

Groups of mice received intraperitoneal injections of 5-10-5 MOE gapmersat a dose of 2.5 mg/kg, 12.5 mg/kg, or 25 mg/kg once per week for 3weeks. One group of mice received subcutaneous injections of PBS onceweekly for 2 weeks. The PBS-injected groups served as the control groupsto which the corresponding oligonucleotide-treated groups were compared.

RNA Analysis

At the end of the treatment period, RNA was extracted from liver forreal-time PCR analysis of measurement of mRNA expression of ANGPTL3 withhANGPTL3_LTS01022 (forward sequence AAATTTTAGCCAATGGCCTCC, designatedherein as SEQ ID NO: 10; reverse sequence TGTCATTAATTTGGCCCTTCG,designated herein as SEQ ID NO: 11; probe sequenceTCAGTTGGGACATGGTCTTAAAGACTTTGTCC, designated herein as SEQ ID NO: 12).Results are presented as percent change of mRNA, relative to PBScontrol, normalized with RIBOGREEN. As shown in the Table below,treatment with ISIS antisense oligonucleotides resulted in significantreduction of ANGPTL3 mRNA in comparison to the PBS control. The ED₅₀ ofeach gapmer is also presented in the Table below. ‘n.d.’ indicates thatthe ED₅₀ could not be determined.

TABLE 174 Percent inhibition of ANGPTL3 mRNA in transgenic mouse liverrelative to the PBS control Dose SEQ ISIS No (mg/kg) % ED₅₀ ID NO 23371025 88 8 233 12.5 79 2.5 0 544145 25 90 4 16 12.5 74 2.5 39 544162 25 539 18 12.5 63 2.5 39 544199 25 81 7 20 12.5 82 2.5 7 560306 25 0 n.d. 3412.5 0 2.5 0 560400 25 87 5 35 12.5 76 2.5 24 560401 25 89 8 36 12.5 622.5 5 560469 25 73 3 38 12.5 78 2.5 50 560735 25 26 31 49 12.5 37 2.5 51567320 25 74 12 93 12.5 37 2.5 32 567321 25 75 11 94 12.5 61 2.5 0

Protein Analysis

Human ANGPTL3 protein levels were quantified using a commerciallyavailable ELISA kit (Catalog #DANL30 by R&D Systems, Minneapolis, Minn.)with transgenic plasma samples diluted 1:20,000 using the manufacturerdescribed protocol. The results are presented in the Table below. Theresults indicate that treatment with ISIS oligonucleotides resulted inreduced ANGPTL3 protein levels. ‘n.d.’ indicates that the ED₅₀ could notbe determined.

TABLE 175 Percent inhibition of plasma protein levels in the transgenicmouse Dose SEQ ISIS No (mg/kg) % ED₅₀ ID NO 233710 25 80 11 233 12.5 562.5 0 544145 25 88 9 16 12.5 64 2.5 0 544162 25 56 15 18 12.5 46 2.5 24544199 25 73 6 20 12.5 73 2.5 31 560306 25 63 n.d. 34 12.5 55 2.5 53560400 25 88 6 35 12.5 73 2.5 20 560401 25 88 10 36 12.5 61 2.5 0 56046925 75 4 38 12.5 70 2.5 52 560735 25 27 34 49 12.5 37 2.5 34 567320 25 6910 93 12.5 44 2.5 39 567321 25 68 12 94 12.5 62 2.5 1

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on day 17, plasma levelsof transaminases (ALT and AST) were measured using an automated clinicalchemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The resultsare presented in the Table below. ISIS oligonucleotides that causedchanges in the levels of any of these liver function markers outside theexpected range for antisense oligonucleotides were excluded in furtherstudies.

TABLE 176 Plasma transaminase levels (IU/L) in transgenic mice on day 17Dose SEQ (mg/kg) ALT AST ID NO PBS — 25 38 ISIS 25 27 40 233 233710 12.524 45 2.5 23 36 ISIS 25 30 56 16 544145 12.5 25 52 2.5 28 43 ISIS 25 2852 18 544162 12.5 36 53 2.5 28 50 ISIS 25 24 47 20 544199 12.5 23 60 2.524 44 ISIS 25 21 45 34 560306 12.5 24 49 2.5 24 47 ISIS 25 22 38 35560400 12.5 21 53 2.5 23 52 ISIS 25 36 80 36 560401 12.5 27 75 2.5 22 49ISIS 25 24 121 38 560469 12.5 23 53 2.5 21 88 ISIS 25 20 48 49 56073512.5 22 138 2.5 24 78 ISIS 25 21 65 93 567320 12.5 20 58 2.5 23 46 ISIS25 23 62 94 567321 12.5 21 49 2.5 24 67

Study 4

Male and female Tg mice were maintained on a 12-hour light/dark cycle.Animals were acclimated for at least 7 days in the research facilitybefore initiation of the experiment. Antisense oligonucleotides (ASOs)were prepared in buffered saline (PBS) and sterilized by filteringthrough a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBSfor injection.

Groups of mice received intraperitoneal injections of 5-10-5 MOE gapmersat a dose of 25 mg/kg once per week for 2 weeks. One group of micereceived subcutaneous injections of PBS once weekly for 2 weeks. ThePBS-injected group served as the control group to which thecorresponding oligonucleotide-treated groups were compared.

RNA Analysis

At the end of the treatment period, RNA was extracted from liver forreal-time PCR analysis of measurement of mRNA expression of ANGPTL3 withhANGPTL3_LTS01022. Results are presented as percent change of mRNA,relative to PBS control, normalized with RIBOGREEN. As shown in theTable below, treatment with ISIS antisense oligonucleotides resulted insignificant reduction of ANGPTL3 mRNA in comparison to the PBS control.

TABLE 177 Percent inhibition of ANGPTL3 mRNA in transgenic mouse liverrelative to the PBS control SEQ ISIS No % ID NO 233710 68 233 544120 6315 544199 82 20 544355 0 21 560268 36 32 560470 47 39 560471 67 40560474 57 41 560566 45 42 560567 68 43 560607 37 46 560608 15 47 56074425 51 560778 32 52 560811 27 54 560925 0 56 563639 5 79 567291 8 91567330 30 95 568049 48 101 568146 26 104

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on day 10, plasma levelsof transaminases (ALT and AST) were measured using an automated clinicalchemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The resultsare presented in the Table below. ISIS oligonucleotides that causedchanges in the levels of any of these liver function markers outside theexpected range for antisense oligonucleotides were excluded in furtherstudies.

TABLE 178 Plasma transaminase levels (IU/L) in transgenic mice on day 10SEQ ALT AST ID NO PBS 29 41 ISIS 233710 29 48 233 ISIS 544120 24 35 15ISIS 544199 27 57 20 ISIS 544355 23 44 21 ISIS 560268 23 42 32 ISIS560470 26 42 39 ISIS 560471 21 50 40 ISIS 560474 20 33 41 ISIS 560566 27102 42 ISIS 560567 20 37 43 ISIS 560607 25 47 46 ISIS 560608 20 49 47ISIS 560744 26 66 51 ISIS 560778 24 87 52 ISIS 560811 21 63 54 ISIS560925 25 115 56 ISIS 563639 20 43 79 ISIS 567291 20 67 91 ISIS 56733029 78 95 ISIS 568049 25 63 101 ISIS 568146 28 140 104

Study 5

Male and female Tg mice were maintained on a 12-hour light/dark cycle.Animals were acclimated for at least 7 days in the research facilitybefore initiation of the experiment. Antisense oligonucleotides (ASOs)were prepared in buffered saline (PBS) and sterilized by filteringthrough a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBSfor injection.

Groups of mice received intraperitoneal injections of 5-10-5 MOE gapmersor deoxy, MOE, and cEt gapmers at a dose of 25 mg/kg once per week for 2weeks. One group of mice received subcutaneous injections of PBS onceweekly for 2 weeks. The PBS-injected group served as the control groupto which the corresponding oligonucleotide-treated groups were compared.

RNA Analysis

At the end of the treatment period, RNA was extracted from liver forreal-time PCR analysis of measurement of mRNA expression of ANGPTL3 withRTS 1984. Results are presented as percent change of mRNA, relative toPBS control, normalized with RIBOGREEN®. As shown in the Table below,treatment with ISIS antisense oligonucleotides resulted in significantreduction of ANGPTL3 mRNA in comparison to the PBS control.

TABLE 179 Percent inhibition of ANGPTL3 mRNA in transgenic mouse liverrelative to the PBS control SEQ ISIS No Chemistry % ID NO 233710 5-10-5MOE 79 233 544156 5-10-5 MOE 92 17 559277 Deoxy, MOE and cEt 75 110560265 5-10-5 MOE 52 31 560285 5-10-5 MOE 42 33 560574 5-10-5 MOE 93 44560847 5-10-5 MOE 61 69 560992 Deoxy, MOE and cEt 80 112 561010 Deoxy,MOE and cEt 66 113 561011 Deoxy, MOE and cEt 96 114 561022 Deoxy, MOEand cEt 79 115 561025 Deoxy, MOE and cEt 57 116 563580 5-10-5 MOE 80 77567115 5-10-5 MOE 78 88 567233 5-10-5 MOE 91 90

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on day 9, plasma levelsof transaminases (ALT and AST) were measured using an automated clinicalchemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The resultsare presented in the Table below. ISIS oligonucleotides that causedchanges in the levels of any of these liver function markers outside theexpected range for antisense oligonucleotides were excluded in furtherstudies.

TABLE 180 Plasma transaminase levels (IU/L) in transgenic mice on day 9SEQ Chemistry ALT AST ID NO PBS — 48 65 ISIS 233710 5-10-5 MOE 24 43 233ISIS 544156 5-10-5 MOE 29 44 17 ISIS 559277 Deoxy, MOE and cEt 22 38 110ISIS 560265 5-10-5 MOE 28 83 31 ISIS 560285 5-10-5 MOE 29 44 33 ISIS560574 5-10-5 MOE 24 54 44 ISIS 560847 5-10-5 MOE 25 45 69 ISIS 560992Deoxy, MOE and cEt 32 128 112 ISIS 561010 Deoxy, MOE and cEt 22 51 113ISIS 561011 Deoxy, MOE and cEt 28 43 114 ISIS 561022 Deoxy, MOE and cEt51 85 115 ISIS 561025 Deoxy, MOE and cEt 22 48 116 ISIS 563580 5-10-5MOE 28 109 77 ISIS 567115 5-10-5 MOE 21 42 88 ISIS 567233 5-10-5 MOE 2273 90

Study 6

Male and female Tg mice were maintained on a 12-hour light/dark cycle.Animals were acclimated for at least 7 days in the research facilitybefore initiation of the experiment. Antisense oligonucleotides (ASOs)were prepared in buffered saline (PBS) and sterilized by filteringthrough a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBSfor injection.

Groups of mice received intraperitoneal injections of deoxy, MOE, andcEt oligonucleotides at a dose of 25 mg/kg once per week for 2 weeks.ISIS 233710, a 5-10-5 MOE gapmer, was also included as a benchmark. Onegroup of mice received subcutaneous injections of PBS once weekly for 2weeks. The PBS-injected group served as the control group to which thecorresponding oligonucleotide-treated groups were compared.

RNA Analysis

At the end of the treatment period, RNA was extracted from liver forreal-time PCR analysis of measurement of mRNA expression of ANGPTL3 withhANGPTL3_LTS01022. Results are presented as percent change of mRNA,relative to PBS control, normalized with RIBOGREEN. As shown in theTable below, treatment with several of the ISIS antisenseoligonucleotides resulted in significant reduction of ANGPTL3 mRNA incomparison to the PBS control.

TABLE 181 Percent inhibition of ANGPTL3 mRNA in transgenic mouse liverrelative to the PBS control SEQ ISIS No Chemistry % ID NO 233710 5-10-5MOE 68 233 561026 Deoxy, MOE and cEt 94 117 561079 Deoxy, MOE and cEt 51160 561084 Deoxy, MOE and cEt 56 161 561123 Deoxy, MOE and cEt 47 163561208 Deoxy, MOE and cEt 42 118 561241 Deoxy, MOE and cEt 13 164 561400Deoxy, MOE and cEt 31 173 561418 Deoxy, MOE and cEt 32 169 561436 Deoxy,MOE and cEt 67 170 561443 Deoxy, MOE and cEt 12 171 561458 Deoxy, MOEand cEt 57 124

Protein Analysis

Human ANGPTL3 protein levels were quantified using a commerciallyavailable ELISA kit (Catalog #DANL30 by R&D Systems, Minneapolis, Minn.)with transgenic plasma samples diluted 1:20,000 using the manufacturerdescribed protocol. The results are presented in the Table below. Theresults indicate that treatment with several of the ISISoligonucleotides resulted in reduced ANGPTL3 protein levels.

TABLE 182 Percent inhibition of plasma protein levels in the transgenicmouse SEQ ID ISIS No Chemistry % NO 233710 5-10-5 MOE 82 233 561026Deoxy, MOE and cEt 92 117 561079 Deoxy, MOE and cEt 80 160 561084 Deoxy,MOE and cEt 89 161 561123 Deoxy, MOE and cEt 62 163 561208 Deoxy, MOEand cEt 0 118 561241 Deoxy, MOE and cEt 36 164 561400 Deoxy, MOE and cEt60 173 561418 Deoxy, MOE and cEt 42 169 561436 Deoxy, MOE and cEt 46 170561443 Deoxy, MOE and cEt 27 171 561458 Deoxy, MOE and cEt 71 124

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on day 10, plasma levelsof transaminases (ALT and AST) were measured using an automated clinicalchemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The resultsare presented in the Table below. ISIS oligonucleotides that causedchanges in the levels of any of these liver function markers outside theexpected range for antisense oligonucleotides were excluded in furtherstudies.

TABLE 183 Plasma transaminase levels (IU/L) in transgenic mice on day 10SEQ ID Chemistry ALT AST NO PBS — 41 64 ISIS 233710 5-10-5 MOE 25 74 233ISIS 561026 Deoxy, MOE and cEt 30 67 117 ISIS 561079 Deoxy, MOE and cEt42 62 160 ISIS 561084 Deoxy, MOE and cEt 70 101 161 ISIS 561123 Deoxy,MOE and cEt 24 41 163 ISIS 561208 Deoxy, MOE and cEt 203 168 118 ISIS561241 Deoxy, MOE and cEt 26 47 164 ISIS 561400 Deoxy, MOE and cEt 27 83173 ISIS 561418 Deoxy, MOE and cEt 58 164 169 ISIS 561436 Deoxy, MOE andcEt 24 42 170 ISIS 561443 Deoxy, MOE and cEt 27 91 171 ISIS 561458Deoxy, MOE and cEt 30 144 124

Study 7

Male and female Tg mice were maintained on a 12-hour light/dark cycle.Animals were acclimated for at least 7 days in the research facilitybefore initiation of the experiment. Antisense oligonucleotides (ASOs)were prepared in buffered saline (PBS) and sterilized by filteringthrough a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBSfor injection.

Groups of mice received intraperitoneal injections of deoxy, MOE, andcEt oligonucleotides at a dose of 25 mg/kg once per week for 2 weeks.ISIS 233710, a 5-10-5 MOE gapmer, was also included as a benchmark. Onegroup of mice received subcutaneous injections of PBS once weekly for 2weeks. The PBS-injected group served as the control group to which thecorresponding oligonucleotide-treated groups were compared.

RNA Analysis

At the end of the treatment period, RNA was extracted from liver forreal-time PCR analysis of measurement of mRNA expression of ANGPTL3 withhANGPTL3_LTS01022. Results are presented as percent change of mRNA,relative to PBS control, normalized with RIBOGREEN®. As shown in theTable below, treatment with ISIS antisense oligonucleotides resulted insignificant reduction of ANGPTL3 mRNA in comparison to the PBS control.

TABLE 184 Percent inhibition of ANGPTL3 mRNA in transgenic mouse liverrelative to the PBS control SEQ ID ISIS No Chemistry % NO 233710 5-10-5MOE 80 233 561462 Deoxy, MOE and cEt 84 126 561463 Deoxy, MOE and cEt 84127 561486 Deoxy, MOE and cEt 74 130 561487 Deoxy, MOE and cEt 82 131561504 Deoxy, MOE and cEt 51 133 561528 Deoxy, MOE and cEt 87 174 561565Deoxy, MOE and cEt 94 175 561566 Deoxy, MOE and cEt 76 176 561571 Deoxy,MOE and cEt 51 178 561621 Deoxy, MOE and cEt 93 134 561646 Deoxy, MOEand cEt 39 140 561649 Deoxy, MOE and cEt 93 141 561650 Deoxy, MOE andcEt 82 142 561689 Deoxy, MOE and cEt 51 180 561722 Deoxy, MOE and cEt 88183 561723 Deoxy, MOE and cEt 85 184 561770 Deoxy, MOE and cEt 70 143562024 Deoxy, MOE and cEt 82 189

Protein Analysis

Human ANGPTL3 protein levels were quantified using a commerciallyavailable ELISA kit (Catalog #DANL30 by R&D Systems, Minneapolis, Minn.)with transgenic plasma samples diluted 1:20,000 using the manufacturerdescribed protocol. The results are presented in the Table below. Theresults indicate that treatment with some of the ISIS oligonucleotidesresulted in reduced ANGPTL3 levels. In this case, ‘0’ value implies thattreatment with the ISIS oligonucleotide did not inhibit expression; insome instances, increased levels of expression may have been recorded.

TABLE 185 Percent inhibition of plasma protein levels in the transgenicmouse SEQ ID ISIS No Chemistry % NO 233710 5-10-5 MOE 60 233 561462Deoxy, MOE and cEt 62 126 561463 Deoxy, MOE and cEt 59 127 561486 Deoxy,MOE and cEt 0 130 561487 Deoxy, MOE and cEt 0 131 561504 Deoxy, MOE andcEt 0 133 561528 Deoxy, MOE and cEt 0 174 561565 Deoxy, MOE and cEt 71175 561566 Deoxy, MOE and cEt 0 176 561571 Deoxy, MOE and cEt 0 178561621 Deoxy, MOE and cEt 72 134 561646 Deoxy, MOE and cEt 0 140 561649Deoxy, MOE and cEt 63 141 561650 Deoxy, MOE and cEt 0 142 561689 Deoxy,MOE and cEt 0 180 561722 Deoxy, MOE and cEt 0 183 561723 Deoxy, MOE andcEt 0 184 561770 Deoxy, MOE and cEt 0 143 562024 Deoxy, MOE and cEt 0189

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on day 9, plasma levelsof transaminases (ALT and AST) were measured using an automated clinicalchemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The resultsare presented in the Table below. ISIS oligonucleotides that causedchanges in the levels of any of these liver function markers outside theexpected range for antisense oligonucleotides were excluded in furtherstudies.

TABLE 186 Plasma transaminase levels (IU/L) in transgenic mice on day 9SEQ ID Chemistry ALT AST NO PBS — 35 72 ISIS 233710 5-10-5 MOE 23 39 233ISIS 561462 Deoxy, MOE and cEt 26 56 126 ISIS 561463 Deoxy, MOE and cEt34 61 127 ISIS 561486 Deoxy, MOE and cEt 23 61 130 ISIS 561487 Deoxy,MOE and cEt 21 64 131 ISIS 561504 Deoxy, MOE and cEt 26 66 133 ISIS561528 Deoxy, MOE and cEt 26 86 174 ISIS 561565 Deoxy, MOE and cEt 24 43175 ISIS 561566 Deoxy, MOE and cEt 23 62 176 ISIS 561571 Deoxy, MOE andcEt 26 68 178 ISIS 561621 Deoxy, MOE and cEt 26 96 134 ISIS 561646Deoxy, MOE and cEt 24 77 140 ISIS 561649 Deoxy, MOE and cEt 22 94 141ISIS 561650 Deoxy, MOE and cEt 34 121 142 ISIS 561689 Deoxy, MOE and cEt24 73 180 ISIS 561722 Deoxy, MOE and cEt 34 89 183 ISIS 561723 Deoxy,MOE and cEt 24 65 184 ISIS 561770 Deoxy, MOE and cEt 22 69 143 ISIS562024 Deoxy, MOE and cEt 32 162 189

Study 8

Male and female Tg mice were maintained on a 12-hour light/dark cycle.Animals were acclimated for at least 7 days in the research facilitybefore initiation of the experiment. Antisense oligonucleotides (ASOs)were prepared in buffered saline (PBS) and sterilized by filteringthrough a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBSfor injection.

Groups of mice received intraperitoneal injections of deoxy, MOE, andcEt oligonucleotides at a dose of 25 mg/kg once per week for 2 weeks.ISIS 233710, a 5-10-5 MOE gapmer, was also included as a benchmark. Onegroup of mice received subcutaneous injections of PBS once weekly for 2weeks. The PBS-injected group served as the control group to which thecorresponding oligonucleotide-treated groups were compared.

RNA Analysis

At the end of the treatment period, RNA was extracted from liver forreal-time PCR analysis of measurement of mRNA expression of ANGPTL3 withhANGPTL3_LTS01022. Results are presented as percent change of mRNA,relative to PBS control, normalized with RIBOGREEN. As shown in theTable below, treatment with ISIS antisense oligonucleotides resulted insignificant reduction of ANGPTL3 mRNA in comparison to the PBS control.

TABLE 187 Percent inhibition of ANGPTL3 mRNA in transgenic mouse liverrelative to the PBS control SEQ ID ISIS No Chemistry % NO 233710 5-10-5MOE 99 233 562078 Deoxy, MOE and cEt 73 147 562086 Deoxy, MOE and cEt 85148 562103 Deoxy, MOE and cEt 58 149 562110 Deoxy, MOE and cEt 94 150562155 Deoxy, MOE and cEt 85 192 562181 Deoxy, MOE and cEt 79 195 562433Deoxy, MOE and cEt 59 155 562436 Deoxy, MOE and cEt 99 156 586669 Deoxy,MOE and cEt 95 210 586676 Deoxy, MOE and cEt 80 211

Protein Analysis

Human ANGPTL3 protein levels were quantified using a commerciallyavailable ELISA kit (Catalog #DANL30 by R&D Systems, Minneapolis, Minn.)with transgenic plasma samples diluted 1:20,000 using the manufacturerdescribed protocol. The results are presented in the Table below. Theresults indicate that treatment with the ISIS oligonucleotides resultedin reduced ANGPTL3 levels.

TABLE 188 Percent inhibition of plasma protein levels in the transgenicmouse SEQ ID ISIS No Chemistry % NO 233710 5-10-5 MOE 69 233 562078Deoxy, MOE and cEt 44 147 562086 Deoxy, MOE and cEt 91 148 562103 Deoxy,MOE and cEt 26 149 562110 Deoxy, MOE and cEt 68 150 562155 Deoxy, MOEand cEt 75 192 562181 Deoxy, MOE and cEt 86 195 562433 Deoxy, MOE andcEt 80 155 562436 Deoxy, MOE and cEt 98 156 586669 Deoxy, MOE and cEt 98210 586676 Deoxy, MOE and cEt 95 211

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on day 8, plasma levelsof transaminases (ALT and AST) were measured using an automated clinicalchemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The resultsare presented in the Table below. ISIS oligonucleotides that causedchanges in the levels of any of these liver function markers outside theexpected range for antisense oligonucleotides were excluded in furtherstudies.

TABLE 189 Plasma transaminase levels (IU/L) in transgenic mice on day 8SEQ ID Chemistry ALT AST NO PBS — 44 248 ISIS 233710 5-10-5 MOE 27 52233 ISIS 562078 Deoxy, MOE and cEt 41 130 147 ISIS 562086 Deoxy, MOE andcEt 30 62 148 ISIS 562103 Deoxy, MOE and cEt 35 99 149 ISIS 562110Deoxy, MOE and cEt 30 161 150 ISIS 562155 Deoxy, MOE and cEt 68 622 192ISIS 562181 Deoxy, MOE and cEt 37 168 195 ISIS 562433 Deoxy, MOE and cEt33 209 155 ISIS 562436 Deoxy, MOE and cEt 30 93 156 ISIS 586669 Deoxy,MOE and cEt 27 141 210 ISIS 586676 Deoxy, MOE and cEt 22 60 211

Study 9

Male and female Tg mice were maintained on a 12-hour light/dark cycle.Animals were acclimated for at least 7 days in the research facilitybefore initiation of the experiment. Antisense oligonucleotides (ASOs)were prepared in buffered saline (PBS) and sterilized by filteringthrough a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBSfor injection.

Groups of mice received intraperitoneal injections of deoxy, MOE, andcEt oligonucleotides at a dose of 25 mg/kg once per week for 2 weeks.ISIS 233710, a 5-10-5 MOE gapmer, was also included as a benchmark. Onegroup of mice received subcutaneous injections of PBS once weekly for 2weeks. The PBS-injected group served as the control group to which thecorresponding oligonucleotide-treated groups were compared.

RNA Analysis

At the end of the treatment period, RNA was extracted from liver forreal-time PCR analysis of measurement of mRNA expression of ANGPTL3 withhANGPTL3_LTS01022. Results are presented as percent change of mRNA,relative to PBS control, normalized with RIBOGREEN. As shown in theTable below, treatment with some of the ISIS antisense oligonucleotidesresulted in significant reduction of ANGPTL3 mRNA in comparison to thePBS control. In this case, ‘0’ value implies that treatment with theISIS oligonucleotide did not inhibit expression; in some instances,increased levels of expression may have been recorded.

TABLE 190 Percent inhibition of ANGPTL3 mRNA in transgenic mouse liverrelative to the PBS control SEQ ID ISIS No Chemistry % NO 233710 5-10-5MOE 84 233 586690 Deoxy, MOE and cEt 45 213 586692 Deoxy, MOE and cEt 45220 586700 Deoxy, MOE and cEt 46 221 586707 Deoxy, MOE and cEt 88 218586708 Deoxy, MOE and cEt 73 222 586718 Deoxy, MOE and cEt 20 219 586744Deoxy, MOE and cEt 0 223 586745 Deoxy, MOE and cEt 0 224 586755 Deoxy,MOE and cEt 75 226 586761 Deoxy, MOE and cEt 66 227 586787 Deoxy, MOEand cEt 47 228 586796 Deoxy, MOE and cEt 88 229 586797 Deoxy, MOE andcEt 81 230 586802 Deoxy, MOE and cEt 33 231 586804 Deoxy, MOE and cEt 60232

Protein Analysis

Human ANGPTL3 protein levels were quantified using a commerciallyavailable ELISA kit (Catalog #DANL30 by R&D Systems, Minneapolis, Minn.)with transgenic plasma samples diluted 1:20,000 using the manufacturerdescribed protocol. The results are presented in the Table below. Theresults indicate that treatment with some of the ISIS oligonucleotidesresulted in reduced ANGPTL3 levels. In this case, ‘0’ value implies thattreatment with the ISIS oligonucleotide did not inhibit expression; insome instances, increased levels of expression may have been recorded.

TABLE 191 Percent inhibition of plasma protein levels in the transgenicmouse SEQ ID ISIS No Chemistry % NO 233710 5-10-5 MOE 80 233 586690Deoxy, MOE and cEt 21 213 586692 Deoxy, MOE and cEt 46 220 586700 Deoxy,MOE and cEt 0 221 586707 Deoxy, MOE and cEt 84 218 586708 Deoxy, MOE andcEt 32 222 586718 Deoxy, MOE and cEt 0 219 586744 Deoxy, MOE and cEt 0223 586745 Deoxy, MOE and cEt 0 224 586755 Deoxy, MOE and cEt 0 226586761 Deoxy, MOE and cEt 0 227 586787 Deoxy, MOE and cEt 0 228 586796Deoxy, MOE and cEt 40 229 586797 Deoxy, MOE and cEt 50 230 586802 Deoxy,MOE and cEt 0 231 586804 Deoxy, MOE and cEt 0 232

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on day 9, plasma levelsof transaminases (ALT and AST) were measured using an automated clinicalchemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The resultsare presented in the Table below. ISIS oligonucleotides that causedchanges in the levels of any of these liver function markers outside theexpected range for antisense oligonucleotides were excluded in furtherstudies.

TABLE 192 Plasma transaminase levels (IU/L) in transgenic mice on day 9SEQ ID Chemistry ALT AST NO PBS — 28 73 ISIS 233710 5-10-5 MOE 22 86 233ISIS 586690 Deoxy, MOE and cEt 42 120 213 ISIS 586692 Deoxy, MOE and cEt22 45 220 ISIS 586700 Deoxy, MOE and cEt 24 84 221 ISIS 586707 Deoxy,MOE and cEt 26 44 218 ISIS 586708 Deoxy, MOE and cEt 22 48 222 ISIS586718 Deoxy, MOE and cEt 22 39 219 ISIS 586744 Deoxy, MOE and cEt 26 83223 ISIS 586745 Deoxy, MOE and cEt 25 56 224 ISIS 586746 Deoxy, MOE andcEt 77 77 225 ISIS 586755 Deoxy, MOE and cEt 28 148 226 ISIS 586761Deoxy, MOE and cEt 36 126 227 ISIS 586787 Deoxy, MOE and cEt 23 88 228ISIS 586796 Deoxy, MOE and cEt 32 148 229 ISIS 586797 Deoxy, MOE and cEt29 151 230 ISIS 586802 Deoxy, MOE and cEt 35 200 231 ISIS 586804 Deoxy,MOE and cEt 24 87 232

Study 10

Male and female Tg mice were maintained on a 12-hour light/dark cycle.Animals were acclimated for at least 7 days in the research facilitybefore initiation of the experiment. Antisense oligonucleotides (ASOs)were prepared in buffered saline (PBS) and sterilized by filteringthrough a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBSfor injection.

Groups of mice received intraperitoneal injections of 5-10-5 MOE gapmersor deoxy, MOE and cEt oligonucleotides at a dose of 5 mg/kg, 12.5 mg/kg,or 25 mg/kg once per week for 2 weeks. One group of mice receivedsubcutaneous injections of PBS once weekly for 2 weeks. The PBS-injectedgroup served as the control group to which the correspondingoligonucleotide-treated groups were compared.

RNA Analysis

At the end of the treatment period, RNA was extracted from liver forreal-time PCR analysis of measurement of mRNA expression of ANGPTL3 withhANGPTL3_LTS01022, and also with RTS3492_MGB. Results are presented aspercent change of mRNA, relative to PBS control, normalized withRIBOGREEN®. As shown in the Table below, treatment with some of the ISISantisense oligonucleotides resulted in reduction of ANGPTL3 mRNA incomparison to the PBS control.

TABLE 193 Percent inhibition of ANGPTL3 mRNA in transgenic mouse liverrelative to the PBS control Dose SEQ ID ISIS No Chemistry (mg/kg)RTS3492_MGB hANGPTL3_LTS01022 NO 233710 5-10-5 MOE 25 0 8 233 12.5 24 225 12 22 544199 5-10-5 MOE 25 63 59 20 12.5 43 43 5 17 24 559277 Deoxy,MOE 25 37 46 110 and cEt 12.5 0 0 5 0 0 560400 5-10-5 MOE 25 45 48 3512.5 36 50 5 0 0 561010 Deoxy, MOE 25 5 37 113 and cEt 12.5 0 6 5 0 0563580 5-10-5 MOE 25 56 59 77 12.5 43 44 5 5 9 567320 5-10-5 MOE 25 4750 93 12.5 0 0 5 0 0 567321 5-10-5 MOE 25 46 32 94 12.5 0 0 5 0 0

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on day 8, plasma levelsof transaminases (ALT and AST) were measured using an automated clinicalchemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The resultsare presented in the Table below. ISIS oligonucleotides that causedchanges in the levels of any of these liver function markers outside theexpected range for antisense oligonucleotides were excluded in furtherstudies.

TABLE 194 Plasma transaminase levels (IU/L) in transgenic mice on day 8Dose SEQ ID Chemistry (mg/kg) ALT AST NO PBS — — 22 82 ISIS 5-10-5 MOE25 21 41 233 233710 12.5 23 66 5 22 118 ISIS 5-10-5 MOE 25 25 47 20544199 12.5 20 40 5 27 43 ISIS Deoxy, MOE 25 21 34 110 559277 and cEt12.5 21 37 2 22 39 ISIS 5-10-5 MOE 25 21 37 35 560400 12.5 20 44 5 24 35ISIS Deoxy, MOE 25 22 48 113 561010 and cEt 12.5 33 64 5 24 41 ISIS5-10-5 MOE 25 21 36 77 563580 12.5 29 81 5 21 59 ISIS 5-10-5 MOE 25 2247 93 567320 12.5 29 58 5 21 70 ISIS 5-10-5 MOE 25 20 50 94 567321 12.524 102 5 19 53

Example 127: Tolerability of Antisense Oligonucleotides Targeting HumanANGPTL3 in CD1 Mice

CD 1 mice (Charles River, Mass.) are a multipurpose mice model,frequently utilized for safety and efficacy testing. The mice weretreated with ISIS antisense oligonucleotides selected from studiesdescribed above and evaluated for changes in the levels of variousplasma chemistry markers.

Study 1

Male CD 1 mice (one animal per treatment group) were injectedintraperitoneally with a single dose of 200 mg/kg of deoxy, MOE, and cEtoligonucleotide. One male CD1 mouse was injected subcutaneously with asingle dose of PBS. Mice were euthanized 48 hours after the last dose,and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on day 4 plasma levelsof transaminases (ALT and AST) were measured using an automated clinicalchemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The resultsare presented in the Table below. ISIS oligonucleotides that causedchanges in the levels of any of these liver function markers outside theexpected range for antisense oligonucleotides were excluded in furtherstudies.

TABLE 195 Plasma transaminase levels in CD1 mice plasma on day 4 SEQ ALTAST ID (IU/L) (IU/L) NO ISIS 559277 29 43 110 ISIS 560990 19 43 111 ISIS560992 21 36 112 ISIS 561010 31 40 113 ISIS 561011 27 32 114 ISIS 56102235 48 115 ISIS 561025 17 28 116 ISIS 561026 31 43 117 ISIS 561208 32 47118 ISIS 561320 25 37 119 ISIS 561343 41 90 120 ISIS 561345 30 45 121ISIS 561347 31 41 122 ISIS 561458 18 38 124 ISIS 561460 42 59 125 ISIS561463 21 33 127 ISIS 561486 17 39 130 ISIS 561487 18 39 131 ISIS 56150424 41 133 ISIS 561621 31 56 134

Body Weights

Body weights were measured one day after the single dose of ISISoligonucleotide, and are presented in the Table below. ISISoligonucleotides that caused any changes in organ weights outside theexpected range for antisense oligonucleotides were excluded from furtherstudies.

TABLE 196 Body weights (g) of CD1 mice after antisense oligonucleotidetreatment SEQ ID Body weight NO ISIS 559277 27 110 ISIS 560990 28 111ISIS 560992 29 112 ISIS 561010 30 113 ISIS 561011 27 114 ISIS 561022 24115 ISIS 561025 28 116 ISIS 561026 27 117 ISIS 561208 29 118 ISIS 56132027 119 ISIS 561343 24 120 ISIS 561345 25 121 ISIS 561347 28 122 ISIS561458 25 124 ISIS 561460 26 125 ISIS 561463 26 127 ISIS 561486 26 130ISIS 561487 27 131 ISIS 561504 26 133 ISIS 561621 27 134Study 2 Male CD 1 mice (one animal per treatment group) were injectedintraperitoneally with a single dose of 200 mg/kg of deoxy, MOE and cEtoligonucleotides. One male CD 1 mouse was injected subcutaneously with asingle dose of PBS. Mice were euthanized 48 hours after the last dose,and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on day 5 plasma levelsof transaminases (ALT and AST) were measured using an automated clinicalchemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The resultsare presented in the Table below. ISIS oligonucleotides that causedchanges in the levels of any of these liver function markers outside theexpected range for antisense oligonucleotides were excluded in furtherstudies.

TABLE 197 Plasma transaminase levels in CD1 mice plasma on day 5 SEQ ALTAST ID (IU/L) (IU/L) NO ISIS 561622 29 64 136 ISIS 561628 17 24 137 ISIS561646 16 34 140 ISIS 561650 32 51 142 ISIS 561079 19 32 160 ISIS 56108424 56 161 ISIS 561241 60 70 164 ISIS 561462 22 54 126 ISIS 561649 56 53141 ISIS 561770 23 39 143 ISIS 561781 20 41 144 ISIS 561918 31 112 146ISIS 562078 15 33 147 ISIS 562086 19 32 148 ISIS 562110 20 41 150 ISIS562415 13 30 154 ISIS 562433 19 35 155 ISIS 562436 21 37 156 ISIS 56244219 34 158

Body Weights

Body weights were measured on day 5 after the single dose of ISISoligonucleotide, and are presented in the Table below. ISISoligonucleotides that caused any changes in organ weights outside theexpected range for antisense oligonucleotides were excluded from furtherstudies.

TABLE 198 Body weights (g) of CD1 mice after antisense oligonucleotidetreatment Body SEQ weights ID NO ISIS 561622 27 136 ISIS 561628 28 137ISIS 561646 29 140 ISIS 561650 30 142 ISIS 561079 27 160 ISIS 561084 24161 ISIS 561241 28 164 ISIS 561462 27 126 ISIS 561649 29 141 ISIS 56177027 143 ISIS 561781 24 144 ISIS 561918 25 146 ISIS 562078 28 147 ISIS562086 25 148 ISIS 562110 26 150 ISIS 562415 26 154 ISIS 562433 26 155ISIS 562436 27 156 ISIS 562442 26 158

Study 3

Male CD1 mice (four animals per treatment group) were injectedintraperitoneally with 100 mg/kg of 5-10-5 MOE gapmers given once a weekfor 6 weeks. One group of 4 male CD1 mice was injected intraperitoneallywith PBS given once a week for 6 weeks. Mice were euthanized 48 hoursafter the last dose, and organs and plasma were harvested for furtheranalysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides, plasma levels ofvarious liver and kidney function markers were measured on day 45 usingan automated clinical chemistry analyzer (Hitachi Olympus AU400e,Melville, N.Y.). The results are presented in the Table below. ISISoligonucleotides that caused changes in the levels of any of thesemarkers outside the expected range for antisense oligonucleotides wereexcluded in further studies.

TABLE 199 Plasma chemistry marker levels in CD1 mice plasma on day 45ALT AST Albumin BUN Creatinine Bilurubin SEQ (IU/L) (IU/L) (g/dL)(mg/dL) (mg/dL) (mg/dL) ID NO PBS 30 55 2.7 26 0.15 0.17 ISIS 5441451146 1081 2.5 29 0.14 0.24 16 ISIS 544199 244 213 2.6 25 0.13 0.15 20ISIS 560400 211 244 2.5 28 0.14 0.14 35 ISIS 560401 212 269 2.4 31 0.140.12 36 ISIS 560469 165 160 2.4 24 0.11 0.14 38 ISIS 567320 141 146 2.725 0.14 0.15 93 ISIS 567321 106 122 2.5 24 0.11 0.13 94

Body Weights

Body weights were measured on day 43, and are presented in the Tablebelow. Kidney, liver and spleen weights were measured at the end of thestudy on day 45. ISIS oligonucleotides that caused any changes in organweights outside the expected range for antisense oligonucleotides wereexcluded from further studies.

TABLE 200 Weights (g) of CD1 mice after antisense oligonucleotidetreatment SEQ ID Body Kidney Liver Spleen NO PBS 39 0.6 2.1 0.1 ISIS544145 30 0.5 1.9 0.1 16 ISIS 544199 42 0.6 2.9 0.3 20 ISIS 560400 400.6 2.8 0.3 35 ISIS 560401 38 0.6 2.7 0.2 36 ISIS 560469 40 0.6 2.7 0.238 ISIS 567320 39 0.6 2.3 0.3 93 ISIS 567321 42 0.6 2.6 0.3 94

Study 4

Male CD1 mice (four animals per treatment group) were injectedintraperitoneally with 50 mg/kg or 100 mg/kg of 5-10-5 MOE gapmers ordeoxy, MOE and cEt oligonucleotides given once a week for 6 weeks. Onegroup of 4 male CD1 mice was injected intraperitoneally with PBS givenonce a week for 6 weeks. Mice were euthanized 48 hours after the lastdose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides, plasma levels ofvarious liver and kidney function markers were measured on day 46 usingan automated clinical chemistry analyzer (Hitachi Olympus AU400e,Melville, N.Y.). The results are presented in the Table below. ISISoligonucleotides that caused changes in the levels of any of thesemarkers outside the expected range for antisense oligonucleotides wereexcluded in further studies.

TABLE 201 Plasma chemistry marker levels in CD1 mice plasma on day 45Dose ALT AST Albumin BUN Creatinine Bilurubin SEQ Chemistry (mg/kg)(IU/L) (IU/L) (g/dL) (mg/dL) (mg/dL) (mg/dL) ID NO PBS — 28 46 2.7 280.13 0.13 ISIS 544156 5-10-5 MOE 100 80 145 2.2 26 0.12 0.10 17 ISIS560574 5-10-5 MOE 100 182 184 2.5 25 0.14 0.15 44 ISIS 561010 Deoxy, MOE50 32 53 2.4 31 0.15 0.12 113 and cEt ISIS 561011 Deoxy, MOE 50 93 1521.8 27 0.15 0.08 114 and cEt ISIS 560580 5-10-5 MOE 100 50 76 2.5 250.12 0.13 237 ISIS 567115 5-10-5 MOE 100 202 304 2.5 19 0.14 0.12 88ISIS 567233 5-10-5 MOE 100 123 145 2.5 24 0.12 0.12 90

Body Weights

Body weights were measured on day 44, and are presented in the Tablebelow. Kidney, liver and spleen weights were measured at the end of thestudy on day 46. ISIS oligonucleotides that caused any changes in organweights outside the expected range for antisense oligonucleotides wereexcluded from further studies.

TABLE 202 Weights (g) of CD1 mice after antisense oligonucleotidetreatment Dose SEQ Chemistry (mg/kg) Body Kidney Liver Spleen ID NO PBS— 38 0.6 2.1 0.2 ISIS 544156 5-10-5 MOE 100 36 0.5 2.2 0.2 17 ISIS560574 5-10-5 MOE 100 40 0.6 2.6 0.4 44 ISIS 561010 Deoxy, MOE 50 39 0.52.2 0.2 113 and cEt ISIS 561011 Deoxy, MOE 50 39 0.6 2.9 0.3 114 and cEtISIS 560580 5-10-5 MOE 100 39 0.5 2.4 0.2 237 ISIS 567115 5-10-5 MOE 10036 0.5 2.2 0.2 88 ISIS 567233 5-10-5 MOE 100 39 0.6 2.2 0.3 90

Study 5

Male CD1 mice (four animals per treatment group) were injectedintraperitoneally with 50 mg/kg of deoxy, MOE and cEt oligonucleotidesgiven once a week for 6 weeks. One group of 4 male CD1 mice was injectedintraperitoneally with PBS given once a week for 6 weeks. Mice wereeuthanized 48 hours after the last dose, and organs and plasma wereharvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides, plasma levels ofvarious liver and kidney function markers were measured on day 43 usingan automated clinical chemistry analyzer (Hitachi Olympus AU400e,Melville, N.Y.). The results are presented in the Table below. ISISoligonucleotides that caused changes in the levels of any of thesemarkers outside the expected range for antisense oligonucleotides wereexcluded in further studies.

TABLE 203 Plasma chemistry marker levels in CD1 mice plasma on day 43ALT AST Albumin BUN Creatinine Bilurubin SEQ (IU/L) (IU/L) (g/dL)(mg/dL) (mg/dL) (mg/dL) ID NO PBS 35 166 2.6 29 0.12 0.32 ISIS 559277 4577 2.5 29 0.13 0.16 110 ISIS 561022 826 802 2.9 29 0.13 0.99 115 ISIS561025 146 183 2.3 28 0.14 0.13 116 ISIS 561026 93 154 2.6 26 0.11 0.16117 ISIS 561079 1943 1511 2.9 28 0.15 0.94 160 ISIS 561084 153 227 2.627 0.12 0.16 161 ISIS 561123 49 90 2.5 31 0.13 0.13 163 ISIS 561436 2957 2.6 25 0.12 0.12 170

Body Weights

Body weights were measured on day 41, and are presented in the Tablebelow. Kidney, liver and spleen weights were measured at the end of thestudy on day 43. ISIS oligonucleotides that caused any changes in organweights outside the expected range for antisense oligonucleotides wereexcluded from further studies.

TABLE 204 Weights (g) of CD1 mice after antisense oligonucleotidetreatment SEQ ID Body Kidney Liver Spleen NO PBS 37 0.5 2.0 0.1 ISIS559277 38 0.6 2.5 0.3 110 ISIS 561022 31 0.4 3.2 0.1 115 ISIS 561025 370.5 2.6 0.2 116 ISIS 561026 39 0.6 2.1 0.2 117 ISIS 561079 42 0.6 4.00.2 160 ISIS 561084 37 0.6 2.4 0.2 161 ISIS 561123 36 0.6 2.2 0.2 163ISIS 561436 41 0.6 2.4 0.2 170

Example 128: Measurement of Viscosity of ISIS Antisense OligonucleotidesTargeting Human ANGPTL3

The viscosity of select antisense oligonucleotides from the studiesdescribed above was measured with the aim of screening out antisenseoligonucleotides which have a viscosity of more than 40 centipoise (cP).Oligonucleotides having a viscosity greater than 40 cP would have lessthan optimal viscosity.

ISIS oligonucleotides (32-35 mg) were weighed into a glass vial, 120 μLof water was added and the antisense oligonucleotide was dissolved intosolution by heating the vial at 50° C. Part (75 μL) of the pre-heatedsample was pipetted to a micro-viscometer (Cambridge). The temperatureof the micro-viscometer was set to 25° C. and the viscosity of thesample was measured. Another part (20 μL) of the pre-heated sample waspipetted into 10 mL of water for UV reading at 260 nM at 85° C. (Cary UVinstrument). The results are presented in the Table below, where theconcentration of each antisense oligonucleotide was 350 mg/ml, andindicate that most of the antisense oligonucleotides solutions areoptimal in their viscosity under the criterion stated above.

TABLE 205 Viscosity of ISIS antisense oligonucleotides targeting humanANGPTL3 ISIS No. Viscosity (cP) SEQ ID NO 233710 14.65 233 337478 13.34235 544145 11.97 16 544162 8.50 18 544199 11.70 20 560306 5.67 34 5604009.26 35 560401 18.11 36 560402 90.67 37 560469 12.04 38 560735 7.49 49567320 9.05 93 567321 9.62 94 567233 6.72 90 563580 16.83 77 56101026.32 113 561011 43.15 114

Example 129: Tolerability of Antisense Oligonucleotides Targeting HumanANGPTL3 in Sprague-Dawley Rats

Sprague-Dawley rats are a multipurpose model used for safety andefficacy evaluations. The rats were treated with ISIS antisenseoligonucleotides from the studies described in the Examples above andevaluated for changes in the levels of various plasma chemistry markers.

Study 1

Male Sprague-Dawley rats were maintained on a 12-hour light/dark cycleand fed ad libitum with Purina normal rat chow, diet 5001. Groups of 4Sprague-Dawley rats each were injected subcutaneously once a week for 6weeks with PBS or with 100 mg/kg of 5-10-5 MOE gapmers. Forty eighthours after the last dose, rats were euthanized and organs and plasmawere harvested for further analysis.

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function,plasma levels of transaminases were measured using an automated clinicalchemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasmalevels of ALT (alanine transaminase) and AST (aspartate transaminase)were measured on day 45 and the results are presented in the Table belowexpressed in IU/L. Plasma levels of bilirubin were also measured usingthe same clinical chemistry analyzer and the results are also presentedin the Table below expressed in mg/dL. ISIS oligonucleotides that causedchanges in the levels of any markers of liver function outside theexpected range for antisense oligonucleotides were excluded in furtherstudies.

TABLE 206 Liver function markers in Sprague-Dawley rats ALT ASTBilirubin SEQ ID (IU/L) (IU/L) (mg/dL) NO PBS 25 65 0.11 ISIS 544145 225407 0.30 16 ISIS 544199 56 102 0.11 20 ISIS 560400 55 175 0.12 35 ISIS560401 89 206 0.13 36 ISIS 560469 227 290 0.15 38 ISIS 567320 55 1720.11 93 ISIS 567321 39 109 0.10 94

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function,plasma levels of blood urea nitrogen (BUN) and creatinine were measuredusing an automated clinical chemistry analyzer (Hitachi Olympus AU400e,Melville, N.Y.). Results are presented in the Table below, expressed inmg/dL. ISIS oligonucleotides that caused changes in the levels of any ofthe kidney function markers outside the expected range for antisenseoligonucleotides were excluded in further studies. Total urine proteinand urine creatinine levels were measured, and the ratio of total urineprotein to creatinine was evaluated. The results are presented in theTable below.

TABLE 207 Kidney function plasma markers (mg/dL) in Sprague-Dawley ratsSEQ ID BUN Creatinine NO PBS 16 0.27 ISIS 544145 53 0.26 16 ISIS 54419924 0.34 20 ISIS 560400 28 0.31 35 ISIS 560401 29 0.28 36 ISIS 560469 230.32 38 ISIS 567320 26 0.35 93 ISIS 567321 24 0.37 94

TABLE 208 Kidney function urine markers in Sprague-Dawley rats TotalCreatinine protein Protein:Creatinine SEQ ID (mg/dL) (mg/dL) ratio NOPBS 59 90 1.5 ISIS 544145 27 2131 84.8 16 ISIS 544199 24 199 8.6 20 ISIS560400 32 176 5.4 35 ISIS 560401 29 521 17.3 36 ISIS 560469 43 351 8.238 ISIS 567320 34 177 5.2 93 ISIS 567321 54 269 5.3 94

Organ Weights

Body weights were measured on day 42 and presented in the Table below.Liver, spleen and kidney weights were measured at the end of the studyon day 45, and are presented in the Table below. ISIS oligonucleotidesthat caused any changes in organ weights outside the expected range forantisense oligonucleotides were excluded from further studies.

TABLE 209 Body and organ weights (g) of Sprague Dawley rats SEQ ID BodyKidney Liver Spleen NO PBS 441 3.3 11.8 0.8 ISIS 544145 240 3.0 11.2 1.716 ISIS 544199 307 2.6 10.3 2.0 20 ISIS 560400 294 2.8 12.3 2.0 35 ISIS560401 281 3.4 11.6 2.3 36 ISIS 560469 316 3.0 11.8 2.0 38 ISIS 567320312 3.1 12.4 2.5 93 ISIS 567321 332 3.3 11.6 2.3 94

Study 2

Male Sprague-Dawley rats were maintained on a 12-hour light/dark cycleand fed ad libitum with Purina normal rat chow, diet 5001. Groups of 4Sprague-Dawley rats each were injected subcutaneously once a week for 6weeks with PBS or with 50 mg/kg or 100 mg/kg of 5-10-5 MOE gapmers ordeoxy, MOE and cEt oligonucleotides. Forty eight hours after the lastdose, rats were euthanized and organs and plasma were harvested forfurther analysis.

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function,plasma levels of transaminases were measured using an automated clinicalchemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasmalevels of ALT (alanine transaminase) and AST (aspartate transaminase)were measured on day 44 and the results are presented in the Table belowexpressed in IU/L. Plasma levels of bilirubin were also measured usingthe same clinical chemistry analyzer and the results are also presentedin the Table below expressed in mg/dL. ISIS oligonucleotides that causedchanges in the levels of any markers of liver function outside theexpected range for antisense oligonucleotides were excluded in furtherstudies.

TABLE 210 Liver function markers in Sprague-Dawley rats Dose ALT ASTBilirubin SEQ Chemistry (mg/kg) (IU/L) (IU/L) (mg/dL) ID NO PBS — — 2263 0.09 ISIS 544156 5-10-5 MOE 100 153 221 0.19 17 ISIS 560574 5-10-5MOE 100 62 128 0.24 44 ISIS 561010 Deoxy, MOE 50 32 99 0.12 113 and cEtISIS 561011 Deoxy, MOE 50 56 100 0.11 114 and cEt ISIS 563580 5-10-5 MOE100 74 89 0.09 77 ISIS 567233 5-10-5 MOE 100 41 136 0.08 90

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function,plasma levels of blood urea nitrogen (BUN) and creatinine were measuredon day 44 using an automated clinical chemistry analyzer (HitachiOlympus AU400e, Melville, N.Y.). Results are presented in the Tablebelow, expressed in mg/dL. ISIS oligonucleotides that caused changes inthe levels of any of the kidney function markers outside the expectedrange for antisense oligonucleotides were excluded in further studies.Total urine protein and urine creatinine levels were measured, and theratio of total urine protein to creatinine was evaluated. The resultsare presented in the Table below.

TABLE 211 Kidney function plasma markers (mg/dL) in Sprague-Dawley ratsDose SEQ ID Chemistry (mg/kg) BUN Creatinine NO PBS — — 18 0.31 ISIS544156 5-10-5 MOE 100 27 0.27 17 ISIS 560574 5-10-5 MOE 100 32 0.24 44ISIS 561010 Deoxy, MOE and 50 24 0.31 113 cEt ISIS 561011 Deoxy, MOE and50 33 0.32 114 cEt ISIS 563580 5-10-5 MOE 100 25 0.20 77 ISIS 5672335-10-5 MOE 100 37 0.23 90

TABLE 212 Kidney function urine markers in Sprague-Dawley rats TotalProtein: Dose Creatinine protein Creatinine SEQ Chemistry (mg/kg)(mg/dL) (mg/dL) ratio ID NO PBS — — 55 66 1.2 ISIS 544156 5-10-5 MOE 10026 166 6.2 17 ISIS 560574 5-10-5 MOE 100 39 276 6.8 44 ISIS 561010Deoxy, MOE 50 54 299 5.6 113 and cEt ISIS 561011 Deoxy, MOE 50 41 52511.7 114 and cEt ISIS 563580 5-10-5 MOE 100 44 338 8.1 77 ISIS 5672335-10-5 MOE 100 46 307 6.4 90

Body weights were measured on day 42 and presented in the Table below.Liver, spleen and kidney weights were measured at the end of the studyon day 44, and are presented in the Table below. ISIS oligonucleotidesthat caused any changes in organ weights outside the expected range forantisense oligonucleotides were excluded from further studies.

TABLE 213 Body and organ weights (g) of Sprague Dawley rats Dose SEQChemistry (mg/kg) Body Kidney Liver Spleen ID NO PBS — — 433 3.1 10.80.6 ISIS 544156 5-10-5 MOE 100 291 2.4 10.6 1.6 17 ISIS 560574 5-10-5MOE 100 315 3.1 10.7 2.1 44 ISIS 561010 Deoxy, MOE 50 386 3.0 11.9 2.1113 and cEt ISIS 561011 Deoxy, MOE 50 324 4.1 12.5 2.4 114 and cEt ISIS563580 5-10-5 MOE 100 358 3.0 12.8 1.5 77 ISIS 567233 5-10-5 MOE 100 2862.9 13.0 2.9 90

Study 3

Male Sprague-Dawley rats were maintained on a 12-hour light/dark cycleand fed ad libitum with Purina normal rat chow, diet 5001. Groups of 4Sprague-Dawley rats each were injected subcutaneously once a week for 6weeks with PBS or with 50 mg/kg of deoxy, MOE and cEt oligonucleotides.Forty eight hours after the last dose, rats were euthanized and organsand plasma were harvested for further analysis.

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function,plasma levels of transaminases were measured using an automated clinicalchemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasmalevels of ALT (alanine transaminase) and AST (aspartate transaminase)were measured on day 44 and the results are presented in the Table belowexpressed in IU/L. Plasma levels of bilirubin were also measured usingthe same clinical chemistry analyzer and the results are also presentedin the Table below expressed in mg/dL. ISIS oligonucleotides that causedchanges in the levels of any markers of liver function outside theexpected range for antisense oligonucleotides were excluded in furtherstudies.

TABLE 214 Liver function markers in Sprague-Dawley rats ALT ASTBilirubin SEQ ID (IU/L) (IU/L) (mg/dL) NO PBS 27 87 0.08 ISIS 559277 36108 0.10 110 ISIS 561025 150 260 0.15 116 ISIS 561026 53 105 0.08 117ISIS 561079 87 196 0.09 160 ISIS 561084 62 177 0.11 161 ISIS 561123 3994 0.07 163 ISIS 561436 64 225 0.13 170

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function,plasma levels of blood urea nitrogen (BUN) and creatinine were measuredon day 44 using an automated clinical chemistry analyzer (HitachiOlympus AU400e, Melville, N.Y.). Results are presented in the Tablebelow, expressed in mg/dL. ISIS oligonucleotides that caused changes inthe levels of any of the kidney function markers outside the expectedrange for antisense oligonucleotides were excluded in further studies.Total urine protein and urine creatinine levels were measured, and theratio of total urine protein to creatinine was evaluated. The resultsare presented in the Table below.

TABLE 215 Kidney function plasma markers (mg/dL) in Sprague-Dawley ratsSEQ ID BUN Creatinine NO PBS 12 0.26 ISIS 559277 16 0.30 110 ISIS 56102524 0.34 116 ISIS 561026 61 0.38 117 ISIS 561079 87 0.67 160 ISIS 56108424 0.35 161 ISIS 561123 16 0.31 163 ISIS 561436 39 0.37 170

TABLE 216 Kidney function urine markers in Sprague-Dawley rats TotalCreatinine protein Protein:Creatinine SEQ ID (mg/dL) (mg/dL) ratio NOPBS 42 77 1.9 ISIS 559277 35 253 7.2 110 ISIS 561025 47 583 14.3 116ISIS 561026 22 1993 111.4 117 ISIS 561079 17 1313 75.5 160 ISIS 56108473 571 7.9 161 ISIS 561123 33 925 29.5 163 ISIS 561436 25 789 36.6 170

Organ Weights

Body weights were measured on day 42 and presented in the table below.Liver, spleen and kidney weights were measured at the end of the studyon day 44, and are presented in the Table below. ISIS oligonucleotidesthat caused any changes in organ weights outside the expected range forantisense oligonucleotides were excluded from further studies.

TABLE 217 Body and organ weights (g) of Sprague Dawley rats SEQ ID BodyKidney Liver Spleen NO PBS 419 3.2 10.7 0.7 ISIS 559277 365 3.5 11.2 1.6110 ISIS 561025 335 3.2 12.8 2.7 116 ISIS 561026 334 4.9 13.9 2.3 117ISIS 561079 302 3.9 9.9 0.9 160 ISIS 561084 317 3.5 12.2 1.9 161 ISIS561123 367 3.3 13.5 1.5 163 ISIS 561436 272 3.1 9.8 2.9 170

Example 130: Effect of ISIS Antisense Oligonucleotides Targeting HumanANGPTL3 in Cynomolgus Monkeys

Cynomolgus monkeys were treated with ISIS antisense oligonucleotidesselected from studies described in the Examples above. Antisenseoligonucleotide efficacy and tolerability, as well as theirpharmacokinetic profile in the liver and kidney, were evaluated.

At the time this study was undertaken, the cynomolgus monkey genomicsequence was not available in the National Center for BiotechnologyInformation (NCBI) database; therefore, cross-reactivity with thecynomolgus monkey gene sequence could not be confirmed. Instead, thesequences of the ISIS antisense oligonucleotides used in the cynomolgusmonkeys was compared to a rhesus monkey sequence for homology. It isexpected that ISIS oligonucleotides with homology to the rhesus monkeysequence are fully cross-reactive with the cynomolgus monkey sequence aswell. The human antisense oligonucleotides tested are cross-reactivewith the rhesus genomic sequence (GENBANK Accession No. NW_001108682.1truncated from nucleotides 3049001 to 3062000, designated herein as SEQID NO: 3). The greater the complementarity between the humanoligonucleotide and the rhesus monkey sequence, the more likely thehuman oligonucleotide can cross-react with the rhesus monkey sequence.The start and stop sites of each oligonucleotide to SEQ ID NO: 3 ispresented in the Table below. “Start site” indicates the 5′-mostnucleotide to which the gapmer is targeted in the rhesus monkey genesequence. ‘Mismatches’ indicates the number of nucleobases in the humanoligonucleotide that are mismatched with the rhesus genomic sequence.

TABLE 218 Antisense oligonucleotides complementary to the rhesus ANGPTL3genomic sequence (SEQ ID NO: 3) Target SEQ Start ID ISIS No SiteMismatches Chemistry NO 563580 9315 2 5-10-5 MOE 77 560400 10052 15-10-5 MOE 35 567320 10232 1 5-10-5 MOE 93 567321 10234 1 5-10-5 MOE 94544199 10653 0 5-10-5 MOE 20 567233 6834 2 5-10-5 MOE 90 561011 3220 1Deoxy, MOE and (S)-cEt 114 559277 3265 0 Deoxy, MOE and (S)-cEt 110

Treatment

Prior to the study, the monkeys were kept in quarantine for at least a30 day period, during which the animals were observed daily for generalhealth. The monkeys were 2-4 years old and weighed between 2 and 4 kg.Nine groups of 5 randomly assigned male cynomolgus monkeys each wereinjected subcutaneously with ISIS oligonucleotide or PBS at four siteson the back in a clockwise rotation (i.e. left, top, right, and bottom),one site per dose. The monkeys were given loading doses of PBS or 40mg/kg of ISIS oligonucleotide every two days for the first week (days 1,3, 5, and 7) and were subsequently dosed once a week for 12 weeks (days14, 21, 28, 35, 42, 49, 56, 63, 70, 77, and 84) with PBS or 40 mg/kg ofISIS oligonucleotide.

During the study period, the monkeys were observed twice daily for signsof illness or distress. Any animal experiencing more than momentary orslight pain or distress due to the treatment, injury or illness wastreated by the veterinary staff with approved analgesics or agents torelieve the pain after consultation with the Study Director. Any animalin poor health or in a possible moribund condition was identified forfurther monitoring and possible euthanasia. For example, one animal inthe ISIS 567321 treatment group was found moribund on day 45 and wasterminated. Scheduled euthanasia of the animals was conducted on day 86(approximately 48 hours after the final dose) by exsanguination afterketamine/xylazine-induced anesthesia and administration of sodiumpentobarbital. The protocols described in the Example were approved bythe Institutional Animal Care and Use Committee (IACUC).

Hepatic Target Reduction RNA Analysis

On day 86, RNA was extracted from liver for real-time PCR analysis ofmeasurement of mRNA expression of ANGPTL3. Results are presented aspercent change of mRNA, relative to PBS control, normalized withRIBOGREEN®. As shown in the Table below, treatment with ISIS antisenseoligonucleotides resulted in significant reduction of ANGPTL3 mRNA incomparison to the PBS control.

Analysis of ANGPTL3 mRNA levels revealed that ISIS 544199 and ISIS559277, which are both fully cross-reactive with the rhesus sequence,significantly reduced expression levels. Other ISIS oligonucleotides,which targeted the monkey sequence with mismatches, were also able toreduce ANGPTL3 mRNA levels.

TABLE 219 Percent inhibition of ANGPTL3 mRNA in the cynomolgus monkeyliver relative to the PBS control SEQ ISIS No % ID NO 563580 62 77560400 59 35 567320 67 93 567321 34 94 544199 88 20 561011 47 114 55927785 110

Protein Analysis

Approximately 1 mL of blood was collected from all available animals atday 85 and placed in tubes containing the potassium salt of EDTA. Theblood samples were placed in ice and centrifuged (3000 rpm for 10 min at4° C.) to obtain plasma.

Human ANGPTL3 protein levels were quantified using a commerciallyavailable ELISA kit (Catalog #DANL30 by R&D Systems, Minneapolis, Minn.)with transgenic plasma samples diluted 1:20,000 using the manufacturerdescribed protocol. The results are presented in the Table below.Analysis of plasman ANGPTL3 revealed that ISIS 563580, 544199 and ISIS559277 reduced protein levels in a sustained manner. Other ISISoligonucleotides were also able to reduce ANGPTL3 levels.

TABLE 220 Plasma protein levels (ng/mL) in the cynomolgus monkey SEQ Day1 Day 3 Day 16 Day 30 Day 44 Day 58 Day 72 Day 86 ID NO PBS 142 113 12275 147 170 130 158 ISIS 563580 113 99 102 46 109 93 82 81 77 ISIS 56040092 107 145 63 170 182 157 178 35 ISIS 567320 87 72 94 56 176 181 134 16693 ISIS 567321 80 84 98 62 156 116 122 112 94 ISIS 544199 114 84 50 3466 56 81 71 20 ISIS 567233 115 111 174 134 162 125 122 109 90 ISIS561011 89 92 111 106 104 100 140 129 114 ISIS 559277 86 62 63 54 77 6468 70 110

Tolerability Studies Body Weight Measurements

To evaluate the effect of ISIS oligonucleotides on the overall health ofthe animals, body and weights were measured and are presented in theTable below. The results indicate that effect of treatment withantisense oligonucleotides on body weights was within the expected rangefor antisense oligonucleotides. Specifically, treatment with ISIS 563580was well tolerated in terms of the body weights of the monkeys.

TABLE 221 Final body weights (g) in cynomolgus monkey SEQ Day 1 Day 14Day 28 Day 35 Day 56 Day 70 Day 84 ID NO PBS 2713 2709 2721 2712 27612754 2779 ISIS 563580 2678 2669 2724 2699 2797 2798 2817 77 ISIS 5604002713 2738 2808 2767 2867 2920 2976 35 ISIS 567320 2682 2707 2741 27312804 2830 2853 93 ISIS 567321 2672 2745 2849 2845 2995 2965 3002 94 ISIS544199 2760 2813 2851 2897 2905 2888 2871 20 ISIS 567233 2657 2668 26502677 2907 2963 2903 90 ISIS 561011 2753 2797 2801 2811 2921 2967 2941114 ISIS 559277 2681 2688 2701 2755 2826 2831 2965 110

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function,blood samples were collected from all the study groups. The bloodsamples were collected from the cephalic, saphenous, or femoral veins,48 hours post-dosing. The monkeys were fasted overnight prior to bloodcollection. Blood was collected in tubes without anticoagulant for serumseparation. The tubes were kept at room temperature for a minimum of 90minutes and then centrifuged (approximately 3,000 rpm for 10 min) toobtain serum. Levels of various liver function markers were measuredusing a Toshiba 200FR NEO chemistry analyzer (Toshiba Co., Japan).Plasma levels of ALT and AST were measured and the results are presentedin the Table below, expressed in IU/L. Bilirubin, a liver functionmarker, was similarly measured and is presented in the Table belowexpressed in mg/dL. The results indicate that most of the antisenseoligonucleotides had no effect on liver function outside the expectedrange for antisense oligonucleotides. Specifically, treatment with ISIS563580 was well tolerated in terms of the liver function in monkeys.

TABLE 222 ALT levels (IU/L) in cynomolgus monkey plasma SEQ Day 1 Day 30Day 58 Day 86 ID NO PBS 47 35 32 46 ISIS 563580 56 55 55 83 77 ISIS560400 50 35 47 68 35 ISIS 567320 72 44 51 106 93 ISIS 567321 53 39 4475 94 ISIS 544199 58 49 51 51 20 ISIS 567233 42 38 47 64 90 ISIS 56101148 35 34 43 114 ISIS 559277 49 45 53 60 110

TABLE 223 AST levels (IU/L) in cynomolgus monkey plasma SEQ Day 1 Day 30Day 58 Day 86 ID NO PBS 76 42 39 60 ISIS 563580 75 56 42 81 77 ISIS560400 85 63 59 99 35 ISIS 567320 104 64 55 153 93 ISIS 567321 83 47 4566 94 ISIS 544199 68 68 70 91 20 ISIS 567233 46 80 66 86 90 ISIS 56101148 39 41 51 114 ISIS 559277 50 56 55 77 110

TABLE 224 Bilirubin levels (mg/dL) in cynomolgus monkey plasma SEQ Day 1Day 30 Day 58 Day 86 ID NO PBS 0.31 0.24 0.20 0.19 ISIS 563580 0.34 0.230.17 0.18 77 ISIS 560400 0.29 0.19 0.14 0.13 35 ISIS 567320 0.38 0.240.16 0.19 93 ISIS 567321 0.35 0.20 0.16 0.17 94 ISIS 544199 0.23 0.160.17 0.15 20 ISIS 567233 0.26 0.17 0.15 0.12 90 ISIS 561011 0.20 0.130.16 0.13 114 ISIS 559277 0.22 0.15 0.16 0.15 110

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function,blood samples were collected from all the study groups. The bloodsamples were collected from the cephalic, saphenous, or femoral veins,48 hours post-dosing. The monkeys were fasted overnight prior to bloodcollection. Blood was collected in tubes without anticoagulant for serumseparation. The tubes were kept at room temperature for a minimum of 90minutes and then centrifuged (approximately 3,000 rpm for 10 min) toobtain serum. Levels of BUN and creatinine were measured using a Toshiba200FR NEO chemistry analyzer (Toshiba Co., Japan). Results are presentedin the Table below, expressed in mg/dL.

The plasma chemistry data indicate that most of the ISISoligonucleotides did not have any effect on the kidney function outsidethe expected range for antisense oligonucleotides. Specifically,treatment with ISIS 563580 was well tolerated in terms of the kidneyfunction of the monkeys.

TABLE 225 Plasma BUN levels (mg/dL) in cynomolgus monkeys SEQ Day 1 Day30 Day 58 Day 86 ID NO PBS 28 28 27 29 ISIS 563580 27 27 25 27 77 ISIS560400 25 24 21 27 35 ISIS 567320 27 28 26 32 93 ISIS 567321 25 24 23 2494 ISIS 544199 23 25 24 23 20 ISIS 567233 23 32 30 29 90 ISIS 561011 2524 23 24 114 ISIS 559277 26 28 24 26 110

TABLE 226 Plasma creatinine levels (mg/dL) in cynomolgus monkeys SEQ Day1 Day 30 Day 58 Day 86 ID NO PBS 0.96 0.95 0.89 0.88 ISIS 563580 0.971.04 0.88 0.85 77 ISIS 560400 0.99 1.00 0.93 0.91 35 ISIS 567320 0.950.94 0.89 0.87 93 ISIS 567321 0.97 0.94 0.89 0.87 94 ISIS 544199 0.860.87 0.88 0.87 20 ISIS 567233 0.89 1.08 1.06 1.00 90 ISIS 561011 0.930.93 0.91 0.90 114 ISIS 559277 0.86 0.91 0.87 0.91 110

Hematology

To evaluate any effect of ISIS oligonucleotides in cynomolgus monkeys onhematologic parameters, blood samples of approximately 0.5 mL of bloodwas collected from each of the available study animals in tubescontaining K₂-EDTA. Samples were analyzed for red blood cell (RBC)count, white blood cells (WBC) count, individual white blood cellcounts, such as that of monocytes, neutrophils, lymphocytes, as well asfor platelet count, hemoglobin content and hematocrit, using an ADVIA120hematology analyzer (Bayer, USA). The data is presented in the Tablesbelow.

The data indicate the oligonucleotides did not cause any changes inhematologic parameters outside the expected range for antisenseoligonucleotides at this dose. Specifically, treatment with ISIS 563580was well tolerated in terms of the hematologic parameters of themonkeys.

TABLE 227 Blood cell counts in cynomolgus monkeys RBC Platelets WBCNeutrophils Lymphocytes Monocytes SEQ (×10⁶/μL) (×10³/μL) (×10³/μL) (%WBC) (% total) (% total) ID NO PBS 5.6 462 12.2 58 39 2 ISIS 563580 5.5394 10.7 52 44 2 77 ISIS 560400 5.7 269 10.2 44 50 3 35 ISIS 567320 5.1329 9.1 51 44 3 93 ISIS 567321 5.3 363 8.9 60 36 2 94 ISIS 544199 5.6316 9.7 34 61 3 20 ISIS 567233 5.0 298 12.1 40 53 4 90 ISIS 561011 5.5356 10.2 33 62 3 114 ISIS 559277 5.1 343 8.3 45 49 3 110

TABLE 228 Hematologic parameters in cynomolgus monkeys Hemoglobin HCTSEQ (g/dL) (%) ID NO PBS 13 43 ISIS 563580 12 40 77 ISIS 560400 12 41 35ISIS 567320 11 38 93 ISIS 567321 12 41 94 ISIS 544199 13 44 20 ISIS567233 11 38 90 ISIS 561011 13 42 114 ISIS 559277 12 40 110

Effect on Pro-Inflammatory Molecules

To evaluate any inflammatory effect of ISIS oligonucleotides incynomolgus monkeys, blood samples were taken for analysis of C-reactiveprotein and C3 levels on day 84 pre-dose. Approximately 1.5 mL of bloodwas collected from each animal and put into tubes without anticoagulantfor serum separation. The tubes were kept at room temperature for aminimum of 90 min and then centrifuged at 3,000 rpm for 10 min at roomtemperature to obtain serum. C-reactive protein (CRP) and complement C3,which serve as markers of inflammation, were measured using a Toshiba200FR NEO chemistry analyzer (Toshiba Co., Japan). The results indicatethat treatment with ISIS 563580 was tolerable in monkeys.

TABLE 229 C-reactive protein levels (mg/L) in cynomolgus monkey plasmaSEQ Day 1 Day 30 Day 58 Day 86 ID NO PBS 3.1 5.5 2.7 4.1 ISIS 563580 2.42.4 4.5 3.9 77 ISIS 560400 3.4 7.5 9.2 14.4 35 ISIS 567320 2.5 1.7 2.54.3 93 ISIS 567321 3.7 3.1 5.5 7.0 94 ISIS 544199 1.2 1.5 8.8 8.1 20ISIS 567233 1.9 12.0 6.8 6.6 90 ISIS 561011 1.7 1.2 2.1 3.7 114 ISIS559277 1.8 2.1 10.9 5.2 110

TABLE 230 C3 levels (mg/dL) in cynomolgus monkey plasma SEQ Pre-dose Day84 ID NO PBS 122 117 ISIS 563580 116 84 77 ISIS 560400 120 105 35 ISIS567320 114 100 93 ISIS 567321 106 93 94 ISIS 544199 113 66 20 ISIS567233 113 63 90 ISIS 561011 115 79 114 ISIS 559277 119 87 110

Measurement of Oligonucleotide Concentration

The concentration of the full-length oligonucleotide was measured. Themethod used is a modification of previously published methods (Leeds etal., 1996; Geary et al., 1999) which consist of a phenol-chloroform(liquid-liquid) extraction followed by a solid phase extraction. Aninternal standard (ISIS 355868, a 27-mer 2′-O-methoxyethyl modifiedphosphorothioate oligonucleotide, GCGTTTGCTCTTCTTCTTGCGTTTTTT,designated herein as SEQ ID NO: 13) was added prior to extraction.Tissue sample concentrations were calculated using calibration curves,with a lower limit of quantitation (LLOQ) of approximately 1.14 μg/g.The results are presented in the Table below, expressed as g/g liver orkidney tissue. The ratio of full-length oligonucleotide concentrationsin the kidney versus the liver was calculated. The ratio of full-lengtholigonucleotide concentrations in the kidney versus the liver aftertreatment with ISIS 563580 was found to be most optimal compared toother compounds assessed.

TABLE 231 Oligonucleotide full length concentration Kidney/Liver SEQISIS No Kidney Liver ratio ID NO 563580 1822 1039 1.8 77 560400 38071375 2.8 35 567320 2547 569 4.5 93 567321 2113 463 4.6 94 544199 1547561 2.8 20 561011 2027 477 4.3 114 559277 2201 508 4.3 110

Example 131: Comparison of Antisense Inhibition of Human ANGPTL3 inhuANGPTL3 Transgenic Mice by ISIS Oligonucleotides Comprising a GalNAcConjugate Group and ISIS Oligonucleotides that do not Comprise a GalNAcConjugate Group

Antisense oligonucleotides comprising GalNAc₃-7_(a) were evaluated incomparison with their unconjugated counterparts for their ability toreduce human ANGPTL3 mRNA transcript in Tg mice. The gapmers, whichtarget SEQ ID NO: 1, are described in the Table below and in Table 121.The symbols of the Backbone Chemistry column are as follows: ‘s’ denotesthioate ester and ‘o’ denotes phosphate ester.

TABLE 232 ISIS oligonucleotides Target SEQ Start Backbone ID ISIS NoSequence Site Conjugate Chemistry NO 563580 GGACATTGCCAGTAATCGCA 1140None sssssssssssssssssss 77 703801 GGACATTGCCAGTAATCGCA 1140 GalNAc₃-7asssssssssssssssssss 77 703802 GGACATTGCCAGTAATCGCA 1140 GalNAc₃-7asoooossssssssssooss 77

Female and male Tg mice were maintained on a 12-hour light/dark cycle.Animals were acclimated for at least 7 days in the research facilitybefore initiation of the experiment. Antisense oligonucleotides (ASOs)were prepared in buffered saline (PBS) and sterilized by filteringthrough a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBSfor injection.

A group of 4 mice received subcutaneous injections of ISIS 563580 atdoses of 5 mg/kg, 10 mg/kg, 15 mg/kg, or 30 mg/kg once per week for 2weeks. Groups of 4 mice each received intraperitoneal injections of ISIS703801 or ISIS 703802 at doses of 0.3 mg/kg, 1 mg/kg, 3 mg/kg, or 10mg/kg once per week for 2 weeks. One group of mice received subcutaneousinjections of PBS once weekly for 2 weeks. The PBS-injected group servedas the control group to which the corresponding oligonucleotide-treatedgroups were compared.

RNA Analysis

At the end of the treatment period, RNA was extracted from liver tissuefor real-time PCR analysis of measurement of mRNA expression of ANGPTL3with human primer probe set hANGPTL3_LTS01022. Results are presented aspercent change of mRNA, relative to PBS control, normalized withRIBOGREEN®. A zero value simply indicates that the antisenseoligonucleotide did not inhibit expression at a measurable level.

The results demonstrate that the conjugated compounds are much morepotent in reducing ANGPTL3 expression than their unconjugatedcounterpart as evident from the percent inhibition and ID₅₀ values. Theconjugated oligonucleotide with mixed backbone chemistry (703802) wasmore potent in inhibiting expression than the conjugated oligonucleotidewith full phosphorothioate backbone chemistry (703801).

TABLE 233 Percent inhibition of ANGPTL3 mRNA in transgenic mouse liverrelative to the PBS control Dose % ID₅₀ ISIS No (mg/kg) inhibition(mg/kg/wk) 563580 30 79 6 15 73 10 72 5 40 703801 10 85 1 3 89 1 54 0.332 703802 10 89 0.3 3 85 1 67 0.3 52

Protein Analysis

Human ANGPTL3 protein levels were quantified using a commerciallyavailable ELISA kit (Catalog #DANL30 by R&D Systems, Minneapolis, Minn.)with transgenic plasma samples diluted 1:20,000 using the manufacturerdescribed protocol. The results are presented in the Table below. A zerovalue simply indicates that the antisense oligonucleotide did notinhibit expression at a measurable level.

The results demonstrate that the conjugated compounds are more potent inreducing ANGPTL3 expression than their unconjugated counterpart asevident from the percent inhibition values. The conjugatedoligonucleotide with mixed backbone chemistry (703802) was more potentin inhibiting expression than the conjugated oligonucleotide with fullphosphorothioate backbone chemistry (703801).

TABLE 234 Percent inhibition of plasma protein levels in the transgenicmouse Dose % ISIS No (mg/kg) inhibition 563580 30 77 15 74 10 75 5 56703801 10 82 3 40 1 0 0.3 0 703802 10 81 3 81 1 64 0.3 66

Example 132: Tolerability of a GalNAc Conjugated AntisenseOligonucleotide Targeting Human ANGPTL3 in CD1 Mice

Male CD1 mice (four animals per treatment group) were injectedsubcutaneously with various doses of ISIS 703802 as described in theTable below for 6 weeks (on days 1, 3, 5, 8, 14, 21, 28, 35 and 42). Onegroup of 4 male CD1 mice was injected subcutaneously with PBS for 6weeks. Mice were euthanized 48 hours after the last dose, and organs andplasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS 703802, plasma levels of various liverand kidney function markers were measured on day 44 using an automatedclinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).The results are presented in the Table below. ISIS 703802 was shown tobe a tolerable compound even at high doses.

TABLE 235 Plasma chemistry marker levels in CD1 mice plasma on day 44ALT AST Albumin BUN Creatinine Bilurubin (IU/L) (IU/L) (g/dL) (mg/dL)(mg/dL) (mg/dL) PBS 34 49 2.6 27 0.15 0.13 ISIS 703802 96 81 2.8 25 0.170.17 50 mg/kg/wk ISIS 703802 54 56 2.7 27 0.16 0.17 20 mg/kg/wk ISIS703802 37 49 2.7 28 0.19 0.15 10 mg/kg/wk ISIS 703802 36 46 2.7 26 0.160.16  5 mg/kg/wk

Body Weights

Body, kidney, liver and spleen weights were measured at the end of thestudy on day 44. ISIS 703802 did not significantly change body and organweights even when administered at high doses.

TABLE 236 Weights (g) of CD1 mice after antisense oligonucleotidetreatment Body Kidney Liver Spleen PBS 42 0.64 2.06 0.12 ISIS 703802 390.53 2.42 0.10 50 mg/kg/wk ISIS 703802 40 0.57 2.29 0.13 20 mg/kg/wkISIS 703802 43 0.66 2.36 0.13 10 mg/kg/wk ISIS 703802 42 0.63 2.38 0.13 5 mg/kg/wk

Example 133: Tolerability of a GalNAc Conjugated AntisenseOligonucleotide Targeting Human ANGPTL3 in Sprague-Dawley Rats

Sprague-Dawley rats are a multipurpose model used for safety andefficacy evaluations. Male Sprague-Dawley rats were maintained on a12-hour light/dark cycle and fed ad libitum with Purina normal rat chow,diet 5001. Groups of 4 Sprague-Dawley rats each were injectedsubcutaneously with various doses of ISIS 703802 as described in theTable below for 6 weeks (on days 1, 3, 5, 8, 14, 21, 28, 35, and 42).One group of 4 rats was injected subcutaneously with PBS for 6 weeks.Rats were euthanized 48 hours after the last dose, and organs and plasmawere harvested for further analysis.

Liver and Kidney Function

To evaluate the effect of ISIS 703802 on hepatic function, plasma levelsof transaminases were measured using an automated clinical chemistryanalyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of ALT(alanine transaminase) and AST (aspartate transaminase) were measured onday 44 and the results are presented in the Table below expressed inIU/L.

To evaluate the effect of ISIS 703802 on renal function, plasma levelsof albumin, blood urea nitrogen (BUN), creatitine and bilirubin weremeasured using the same clinical chemistry analyzer and the results arepresented in the Table below expressed in g/dL or mg/dL.

To further evaluate the effect of ISIS 703802 on renal function, urineprotein and urine creatinine levels were measured, and the ratio oftotal urine protein to creatinine was evaluated. The results arepresented in the Table below.

ISIS 703802 was shown to be a tolerable compound even at high doses.

TABLE 237 Liver and kidney function markers in Sprague-Dawley rat plasmaon day 44 ALT AST Albumin BUN Creatinine Bilurubin (IU/L) (IU/L) (g/dL)(mg/dL) (mg/dL) (mg/dL) PBS 28 72 3.2 15 0.25 0.07 ISIS 703802 86 97 3.417 0.26 0.09 50 mg/kg/wk ISIS 703802 62 91 3.3 18 0.29 0.09 20 mg/kg/wkISIS 703802 64 99 3.2 15 0.27 0.08 10 mg/kg/wk ISIS 703802 48 88 3.3 150.26 0.07  5 mg/kg/wk

TABLE 238 Kidney function urine markers (mg/dL) in Sprague-Dawley rat onday 44 Creatinine MTP Protein:Creatinine (mg/dL) (mg/dL) ratio PBS 91100 1.13 ISIS 703802 82 172 2.04 50 mg/kg/wk ISIS 703802 89 178 2.05 20mg/kg/wk ISIS 703802 85 103 1.26 10 mg/kg/wk ISIS 703802 117 134 1.17  5mg/kg/wk

Organ Weights

Body, liver, spleen and kidney weights were measured at the end of thestudy on day 44 and are presented in the Table below. ISIS 703802 didnot significantly change body, kidney and liver weights even whenadministered at high doses.

TABLE 239 Body and organ weights (g) of Sprague Dawley rats Body KidneyLiver Spleen PBS 471 3.6 13 0.67 ISIS 703802 445 3.6 14 1.37 50 mg/kg/wkISIS 703802 435 3.3 14 0.97 20 mg/kg/wk ISIS 703802 464 3.5 14 0.91 10mg/kg/wk ISIS 703802 468 3.0 15 0.75  5 mg/kg/wk

1.-26. (canceled)
 27. A compound comprising a single stranded modifiedoligonucleotide and a conjugate group, wherein the modifiedoligonucleotide consists of 12 to 30 linked nucleosides and comprises anucleobase sequence comprising a portion of at least 8 contiguousnucleobases complementary to an equal length portion of nucleobases 1140to 1159 of SEQ ID NO: 1, wherein the nucleobase sequence of the modifiedoligonucleotide is at least 80% complementary to SEQ ID NO: 1; whereinthe conjugate group is linked to the modified oligonucleotide at the 5′end of the modified oligonucleotide by a conjugate linker; and whereinthe conjugate group comprises:

wherein each n is, independently, from 1 to
 20. 28. The compound ofclaim 27, wherein the nucleobase sequence of the modifiedoligonucleotide is at least 85%, at least 90%, at least 95%, or 100%complementary to SEQ ID NOs:
 1. 29. The compound of claim 27, whereinthe modified oligonucleotide comprises at least one modifiedinternucleoside linkage.
 30. The compound of claim 29, wherein themodified internucleoside linkage is a phosphorothioate internucleosidelinkage.
 31. The compound of claim 27, wherein the modifiedoligonucleotide comprises at least one modified sugar.
 32. The compoundof claim 31, wherein at least one modified sugar is selected from abicyclic sugar, a 2′-O-methoxyethyl modified sugar, a constrained ethylmodified sugar, a 3′-fluoro-HNA or a 4′-(CH₂)_(n)—O-2′ bridge, wherein nis 1 or
 2. 33. The compound of claim 27, wherein at least one nucleosidecomprises a modified nucleobase.
 34. The compound of claim 33, whereinthe modified nucleobase is a 5-methylcytosine.
 35. The compound of claim27, wherein the modified oligonucleotide comprises: a gap segmentconsisting of linked deoxynucleosides; a 5′ wing segment consisting oflinked nucleosides; a 3′ wing segment consisting of linked nucleosides;wherein the gap segment is positioned between the 5′ wing segment andthe 3′ wing segment and wherein each nucleoside of each wing segmentcomprises a modified sugar.
 36. The compound claim 27, wherein themodified oligonucleotide comprises: a gap segment consisting of tenlinked deoxynucleosides; a 5′ wing segment consisting of five linkednucleosides; a 3′ wing segment consisting of five linked nucleosides;wherein the gap segment is positioned between the 5′ wing segment andthe 3′ wing segment, wherein each nucleoside of each wing segmentcomprises a 2′-O-methoxyethyl sugar, and wherein each cytosine residueis a 5-methylcytosine.
 37. The compound of claim 27, wherein eachinternucleoside linkage of the modified oligonucleotide is aphophorothioate linkage.
 38. The compound of claim 36, wherein themodified oligonucleotide consists of 20 linked nucleosides.
 39. Thecompound of claim 27, wherein the conjugate linker has a structureselected from among:

wherein each L is, independently, a phosphorus linking group or aneutral linking group; and each n is, independently, from 1 to
 20. 40.The compound of claim 27, wherein the conjugate linker has a structureselected from among:


41. The compound of claim 27, wherein the conjugate linker has thefollowing structure:


42. The compound of claim 27, wherein the conjugate linker has thefollowing structure:


43. The compound of claim 27, wherein the conjugate linker has astructure selected from among:


44. The compound of claim 27, wherein the conjugate linker has astructure selected from among:


45. The compound of claim 27, wherein the conjugate linker has astructure selected from among:


46. The compound of claim 27, wherein the conjugate linker has astructure selected from among:

wherein each n is, independently, is from 1 to 20; and p is from 1 to 6.47. The compound of claim 27, wherein the conjugate linker has astructure selected from among:

wherein each n is, independently, from 1 to
 20. 48. The compound ofclaim 27, wherein the conjugate linker has a structure selected fromamong:


49. The compound of claim 27, wherein the conjugate linker has astructure selected from among:

wherein n is from 1 to
 20. 50. The compound of claim 27, wherein theconjugate linker has a structure selected from among:


51. The compound of claim 27, wherein the conjugate linker has astructure selected from among:

wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or
 7. 52. Thecompound of claim 27, wherein the conjugate linker has the followingstructure:


53. The compound of claim 27, wherein the conjugate group comprises:


54. The compound of claim 27, wherein the conjugate group comprises:

and the conjugate linker has the following structure:


55. The compound of claim 27, wherein the conjugate group comprises acleavable moiety selected from a phosphodiester, an amide, or an ester.56. The compound of claim 27, wherein the conjugate comprises:


57. A pharmaceutical composition comprising the compound of claim 27 anda pharmaceutically acceptable carrier or diluent.
 58. A methodcomprising administering to an animal the composition of claim
 57. 59.The method of claim 58 wherein the animal is a human.
 60. The method ofclaim 58, for use in treating, preventing, slowing progression of orameliorating a cardiovascular and/or metabolic disease, disorder orcondition.
 61. The method of claim 60, wherein the disease, disorder orcondition is a atherosclerosis, hepatic steatosis, obesity, diabetes,dyslipidemia, coronary heart disease, non-alcoholic fatty liver disease(NAFLD), hyperfattyacidemia and/or metabolic syndrome.
 62. The method ofclaim 61, wherein the hepatic steatosis is nonalcoholic steatohepatitis(NASH) and the dyslipidemia is hyperlipidemia, hypercholesterolemiaand/or hypertriglyceridemia.